SINGLE-USE FLUORESCENT ASSAYS FOR DETERMINATION OF ANALYTES

CROSS-REFERENCED APPLICATIONS

[01] This application claims the priority benefit of U.S. Provisional Application No. 60/704,143, entitled SINGLE-USE FLUORESCENT ASSAYS FOR DETERMINATION OF ANALYTES, which was filed on July 29, 2005 by Fornari et al., and U.S. Provisional Application No. 60/704,252, entitled, SINGLE-USE FLORESCENT ASSAYS FOR DETERMINATION OF ANALYTES, filed on August 1, 2005 by Fornari et al.

BACKGROUND

[02] Traditional fluorescent assays, including, e.g., clinical and forensic laboratory assays often utilize fluorescence or colorimetric reporter constructs for the determination of biomolecular analytes, reporter constructs formed of fluorophores and antibodies for the analyte. Such reporter constructs are used in a signaling system to indicate results of the assay, e.g., the presence or amount of analyte in a sample fluid. Desirable properties in these approaches include, in at least certain applications, one or more of the advantages of rapid result delivery, good sensitivity and specificity, cost effectiveness, ease of use, ability to quantify the analyte(s) of interest, reagent stability, linearity in the clinically reportable range, and good accuracy and reproducibility. Disadvantages in some or all such applications, however, include peak shouldering and tailing in the fluorescence response leading to possible ambiguity or inaccuracy in analyte determination and hook effects at high analyte concentrations. In fluorescent assays for the determination of biomolecular analytes,

disadvantages may include antibody cross reactivity in both single and multi-analyte assays, lack of linearity in the clinically reportable range, and the nonexistence of a sensitive and/or specific antibody for certain analytes. Further, the possibility of determining multiple analytes with sufficient accuracy and reproducibility in a single reaction vessel or by a single analytical test device is in some cases compromised or impossible using current methods and devices, leading to increased complexity, testing expense and/or performance time.

[03] It is an object of at least certain embodiments of the new systems disclosed here, including methods and devices, to provide improved fluorescence assays for determination (i.e., detection and/or quantification) of analytes. Certain embodiments of such new methods and devices reduce or eliminate one or more of the above- mentioned disadvantages of prior known assays. hi accordance with certain embodiments of such new methods and devices, rapid clinical assays are provided for a single analyte or for determination, in some embodiments simultaneous determination, of multiple analytes with a single-use device, in some embodiments at a single test site, e.g., in a single reaction vessel or the like. In accordance with certain embodiments, biomolecular analytes are determined. In accordance with certain embodiments, rapid clinical/environmental/forensic assays are provided for a single analyte or for simultaneous determination of multiple analytes at a single test site. Additional aspects and features of the new systems as well as additional or alternative objects of some or all embodiments of the invention will be understood by

those skilled in this field of technology in view of the following disclosure and in view of the description provided of certain exemplary embodiments.

SUMMARY

[04] In accordance with one aspect, a single-use fluorescence-based assay system, such as a device or method, is provided for determining at least one analyte in a fluid sample. The single-use assay system optionally comprises a disposable or reusable housing or mounting with a single-use assay component operative to receive sample fluid (the sample optionally having first been treated with buffer, diluent, etc.) and perform an assay for a single analyte of interest (sometimes referred to here as the target analyte) or in multiplexed embodiments to perform assays of multiple target analytes. In certain exemplary reusable embodiments the single-use assay component is removable from the housing and replaceable with a fresh assay component operative to perform the same or a different assay (or set of assays). The assay component comprises, for example, one or more reaction chambers along one or more parallel or serial capillary fluid channels, one or more reaction zones in series or in parallel along one or more bibulous strips or pads, etc. Nanocrystals, sometimes referred to as quantum dots or by other terms, are employed in the fluorescent signal system of the single-use assay systems disclosed here. In certain exemplary embodiments the quantum dots have ON basal fluorescent signals, that is, in the natural (unquenched) state they are intrinsically or inherently fluorescent when exposed to excitation light, that is, light of a suitable wavelength, e.g., light from a laser source. In certain exemplary embodiments nanocrystals are employed having a narrow fluorescence peak, that is, nanocrystals that fluoresce strongly in a narrow wavelength band well separated from the wavelength of the excitation light. Certain exemplary embodiments of the systems disclosed here are multiplexed assays employing different quantum dots, that is, quantum dots having different fluorescence peaks, for

different analytes to be assayed in a sample. By detecting the different fluorescence peaks multiple analytes can de determined by the same assay system using a single sample. Such multiplexed assays are said here to determine the multiple target analytes simultaneously, regardless of whether the different analytes are determined in series or in parallel by the assay component, since they are determined from the same sample by the same assay component, even if the analytes are determined at spatially separated reaction zones and even if the sample does not arrive simultaneously at each such site.

[05] In certain exemplary embodiments the quantum dots reagent (also referred to as the nanocrystal reagent) is initially quenched, i.e., the quantum dots employed in such assay reagent or reactant are initially quenched, whereby they are not fluorescent upon exposure to their excitation light (meaning they initially are not substantially fluorescent or are not detectably fluorescent or are not fluorescent above a certain level or degree under the test conditions). Such quenched quantum dots are unquenched and rendered fluorescent in the course of the assay if the target analyte is present (meaning present in sufficient quantity to be detected by the test). In certain alternative embodiments, the quenched quantum dots are unquenched and rendered fluorescent in the course of the assay if the target analyte is not present.

[06] In certain exemplary embodiments the quantum dots reagent is initially unquenched, i.e., the quantum dots employed in such assay reagent are initially unquenched, such that they are fluorescent upon exposure to their excitation light (meaning they initially are substantially fluorescent or detectably fluorescent or fluorescent above a certain level or degree under the test conditions). Such unquenched quantum dots are quenched and rendered fluorescent in the course of the assay if the target analyte is

present or, in certain alternative embodiments, are quenched and rendered fluorescent in the course of the assay if the target analyte is not present. Exemplary of such last mentioned embodiments are competition immunoassays in accordance with the present disclosure employing a first reagent comprising unquenched quantum dots conjugated to antibodies or other suitable affinity or binding sites, e.g., Fab fragments, aptamers or other oligomers or the like (for convenience all referred to here generally as binding sites) for the target analyte and a second reagent comprising a quenching agent, e.g., colloidal gold or the like, conjugated to the target analyte. There may be one or more than one binding site per quantum dot. Upon adding saliva or other biological fluid suspected of containing the target analyte (e.g., a disease marker, a drug of abuse or a metabolite thereof, etc.), the second reagent competes for the binding sites with any target analyte present in the sample fluid. If the target analyte is not present in the sample fluid, the second reagent will bind with and so quench the quantum dots reagent. In such embodiments, therefore, lack of fluorescence upon subsequent exposure to activating radiation indicates that the target analyte was not present in the sample. Numerous suitable alternative versions of these various embodiments will be readily apparent to those skilled in the art given the benefit of this disclosure.

[07] The reagents used in assays according to the present disclosure, e.g., the immediately above-mentioned first and second reagents of embodiments wherein unquenched quantum dots are quenched and rendered fluorescent if the target analyte is not present in the sample fluid, can be used, optionally in conjunction with other reagents, solvating or carrier fluids, etc., in a liquid (e.g., aqueous) system, a lateral flow system, or other suitable system. In lateral flow assay embodiments, for example, the

quantum dots reagent can be immobilized at a detection site on a bibulous strip. The quenching reagent can be provided at a saliva (or other sample fluid) application site or between there and the detection site to be mobilized and carried by the sample fluid (along with any target analyte contained in the sample fluid) to the detection site for competitive binding with the quantum dots reagent. In alternative lateral flow assay embodiments, both the quenching reagent and the quantum dots reagent can be provided at a saliva (or other sample fluid) application site or between there and the detection site, to be mobilized and carried by the sample fluid (along with any target analyte contained in the sample fluid) to the detection site. In other alternative lateral flow assay embodiments, both the quenching reagent and the quantum dots reagent can be provided at the detection site, to be mobilized by the sample fluid. In certain exemplary fluid assay embodiments the quantum dots reagent can be provided in an aqueous or other fluid in a reaction chamber and the quenching reagent can be carried into the reaction chamber with the fluid sample or separately. Numerous suitable alternative arrangements for providing the immunoassay reagents will be readily apparent to those skilled in the art given the benefit of this disclosure.

[08] Certain exemplary embodiments of the single-use assay systems disclosed here optionally comprise a disposable or reusable fluorometer operative to generate excitation illumination of a detection site in the system. Optionally the detection site is at the same location as the reaction site(s) of the system or some other site at which the quantum dots are present (quenched or unquenched depending on whether the analyte of interest was present in the sample) during the fluorescence detection portion of the assay.

[09] In certain exemplary embodiments of the single-use assay system disclosed here, a collection device is provided for collecting the saliva or other biological fluid sample. Such collection device may comprise a wick, capillary or other suitable feature, preferably operative to deliver a predetermined amount of fluid to the reaction chamber or site. Optionally, the collection device is a one-use, disposable device configured to be used with a reusable detector, e.g., a bench-top or hand-held fluorometer, and then discarded.

[10] Certain exemplary embodiments of the single-use assay systems disclosed here optionally further comprise additional disposable or reusable components, functions, features and the like. For example, certain exemplary embodiments may further comprise a display for the assay results, e.g., an observation window operative to provide observation of fluorescence (if any) at a detection site in the housing, and/or a sensor operative to generate a signal corresponding to the presence or absence of fluorescence at the detection site, and/or a power source, such as a battery, a power feed port to receive power from a remote source, etc., and/or reagent reservoirs, and/or an electronic display operative to display results of the assay, and/or electronic communication, memory storage such as a flash chip or other RAM or ROM memory, circuitry for timing the assay and/or otherwise controlling, monitoring, failsafe checking, etc. Certain exemplary embodiments comprise a reusable housing with a reusable fluorometer housed by the housing and a single-use, replaceable assay insert received at or into a socket, docking location or other housing location. The assay insert in such embodiments can comprise a sample receiving site, fluid collection device, the aforesaid reaction site, detection site, etc. Sample may or may not travel in or traverse from one site to another of the assay insert. Thus, for example, the

assay insert may comprise one or more tubes, cells, vials or the like operative to transport sample via capillary action, gravity, etc. The assay insert may, for example, comprise one or more bibulous strips or pads operative to transport sample via absorption (meaning any manner of absorption, adsorption or the like) from site to site. Optionally, some or all of the sample sites in certain exemplary embodiments of the assay insert (sample receiving site, reaction site, detection site, etc.) are at the same location. Thus, for example, sample may be received into a vial pre-charged or subsequently charged with assay reagents, such as the quantum dot reporter construct mentioned above, buffer, a reagent competitive with the analyte of interest, etc. in accordance with known assay techniques whose applicability to the systems disclosed here will be readily apparent to those skilled in the art given the advantage of this disclosure. And, for example, sample may be received onto a bibulous strip pre- charged or subsequently charged with such assay reagents.

[11] hi accordance with another aspect, fluorescence-based systems, such as devices and methods, are provided for determining the presence of at least one analyte in a fluid sample by competitive immunoassay. Assay systems in accordance with this aspect of the disclosure comprise at least the assay component and in certain exemplary embodiments also a disposable or reusable housing having a socket, dock or other site for one or more such single-use assay components and optionally also a disposable or reusable fluorometer housed in the housing. The assay component may be replaceable or the entire assay device may be disposable, that is, single-use. For each target analyte to be determined by the assay system, a reporter construct or signal system of the assay component comprises fluorescent nanocrystals as a fluorochrome quenched by association with quenching conjugate. Suitable nanocrystals are

commercially available or can be produced using known techniques, e.g., Evidot® fluorescent nanocrystals available from Evident Technologies (Troy, New York). The quenching conjugate comprises a competitor to the target analyte conjugated with a quenching moiety, e.g., colloidal gold or other suitable quenching moiety effective under the assay condition to quench the fluorescence of the nanocrystals in the absence of the target analyte. The competitor to the target analyte may be the target analyte itself, a suitable synthetic or natural analog thereof, or other suitable substitute for the target analyte. In operation of the assay, if target analyte is present in the sample it will displace some or all of the quenching conjugate, e.g., at the reaction site of the assay component, thus at least partly unquenching the associated nanocrystals and so rendering them detectably fluorescent. It will be within the ability of those skilled in the art to determine and select suitable reagent specificities, reactant quantities etc. for qualitative and quantitative embodiments of such assays disclosed here.

[12] Certain exemplary solution-based embodiments of the assay systems disclosed immediately above comprise beads, such as micro-beads, incorporating fluorescent nanocrystals that (when unquenched) fluoresce at a first characteristic wavelength band on exposure to excitation radiation. The micro-beads can be formed of any of numerous suitable materials including, for example, agarose, latex, polystyrene, etc. The beads can vary in size from a fraction of a micron to multiple microns in diameter, for example, approximately 1 micron in diameter. The number of fluorescent nanocrystals incorporated into each bead can vary from ten or fewer to as many as thousands of nanocrystals per bead. The nanocrystals may be incorporated into the bead in any suitable manner, e.g., during manufacture of the bead or by

incorporation into the bead after it is formed, etc. The nanocrystals may be incorporated by impregnation, e.g., into or onto the bead, encapsulating the bead, etc. Antibodies or Fab fragments or other suitable affinity or binding sites (for convenience all referred to here generally as binding sites) for the target analyte are associated with the bead, e.g., as a film on the surface of the bead or, e.g., by layering or coating the bead with such antibodies or Fab fragments or the like, or impregnating or otherwise incorporating the antibodies or Fab fragments onto or into the surface of the bead. Conjugates of the target analyte (or a suitable analog, or other substitute for the target analyte that binds to the antibodies or Fab fragments of the bead) and a quenching moiety, e.g., colloidal gold, are bound to the antibodies or Fab fragments. The presence of the conjugates renders the beads (i.e., the nanocrystals thereof) not substantially fluorescent when exposed to the excitation light under assay conditions. Upon exposure to the target analyte in a sample fluid, however, some or all of the conjugates are displaced, thus rendering the beads detectably fluorescent. Displacement may be based, for example, on differential affinity and/or establishment of equilibrium between the conjugates and the target analyte (if any) in the sample. It will be within the ability of those skilled in the art, given the advantage of this disclosure, to establish the displacement properties of the conjugates by the target analyte under assay conditions, whereby the fluorescence of the beads can be correlated qualitatively to the presence or absence of the target analyte in the sample (or to the presence or absence of the target analyte above a threshold quantity) or quantitatively to the amount of target analyte in the sample. A pre-selected quantity of sample may be used (with or without buffering or other treatment) in certain exemplary qualitative or quantitative embodiments.

[13] In certain exemplary multiplexed embodiments of the solution-based assays disclosed immediately above, the assay solution may have, in addition to any other assay reagents, buffers, etc., as described above, a quantity of first beads incorporating nanocrystals that fluoresce at a first characteristic wavelength band, preferably a narrow band, and also a quantity of second beads incorporating nanocrystals that fluoresce at a second wavelength band distinguishable by a fluorometer from the first wavelength band. The second beads can be operative to qualitatively or quantitatively determine a second target analyte in the sample in the same manner as the first beads determine the first target analyte. Likewise, quantities of third beads, fourth beads, etc. can be used for third and fourth analytes to be determined by the assay, and so on.

[14] The beads may be formed of any suitable material, e.g., polymeric, ceramic or other material. In certain exemplary embodiments the beads are substantially inert under assay conditions to the reagents and sample fluids used in the assay. The beads can be pre-charged to the assay component, that is, added to the assay component of the assay system just prior to conducting the assay or well before, e.g., at the time of original manufacture of the assay component. As with other embodiments of the assay systems disclosed here, the assay component of such solution-based embodiments may be pre-loaded into a hand-held housing incorporating a fluorometer and/or other components of the device, such as timing circuitry, communication ports for data output such as the assay results and in some embodiments for data input, a CPU such as a microprocessor, RAM or ROM or other electronic memory, a user interface for the user to enter information about the assay or the sample source such as a graphical user interface, keyboard, touch screen etc., a display device for displaying assay results or other information, etc. The beads can be provided, optionally together

with other reagents (if any) of the assay in a dry or lyophilized condition, and put into the liquid assay medium immediately prior to conducting the assay. In certain exemplary embodiments introducing the sample (treated or untreated) puts the beads into solution. It will be understood by those skilled in the art that at least certain exemplary embodiments of the solution-based assays disclosed here, as for other embodiments, may be FRET-based systems.

[15] Certain exemplary lateral flow embodiments of the assay systems disclosed above comprise an assay component incorporating one or more porous or absorbent or bibulous substrates (for convenience all referred to here as bibulous), e.g., in the shape or form of pads, strips or the like, wherein one or more of such bibulous substrates has fluorescent nanocrystals at one or more reaction sites. Fluid sample is added to the bibulous substrate at a sample addition site. As noted above generally for the assay systems disclosed here, the reaction site may be at the same location as other sites of the assay component, such as the sample addition site, the detection site, etc. The nanocrystals, e.g., Evidot® nanocrystals available from Evident Technology (Troy, New York) fluoresce (when unquenched) on exposure to excitation radiation, e.g., laser light of the appropriate wavelength provided by a fluorometer optionally provided as a component of the assay system. The nanocrystals may be incorporated into the bibulous substrate in any suitable manner, e.g., during manufacture of the bibulous substrate or by incorporation into the bibulous substrate after it is formed, etc. The nanocrystals may be incorporated by impregnation, e.g., into or onto the bibulous substrate, etc. The nanocrystals are quenched by quenching conjugates bound to antibodies or Fab fragments or other suitable affinity or binding sites (for convenience, as noted above, all referred to here generally as binding sites) for the

target analyte. The antibodies or Fab fragments or the like are provided optionally as a film or layer etc. at the reaction site, e.g., a layer or coating on the bibulous substrate of such antibodies or Fab fragments or the like overlying or intimately intermingled with the nanocrystals. The antibodies or Fab fragments are bound to the bibulous substrate in any suitable manner, various of which are well known and will be apparent to those skilled in the art given the benefit of the present disclosure. The quenching conjugates comprise the target analyte (or a suitable analog or other substitute for the target analyte that binds to the antibodies or Fab fragments) and a quenching moiety, e.g., colloidal gold. The quenching conjugates are bound to the antibodies or Fab fragments. The presence of the conjugates renders the nanocrystals not substantially fluorescent when exposed to the excitation light under assay conditions. Upon exposure to the target analyte in a sample fluid, however, some or all of the conjugates are displaced by the target analyte and in certain exemplary embodiments are transported away from the nanocrystals by a flow of sample fluid. In certain exemplary embodiments the quenching conjugates are transported downstream from the reaction site along a generally linear bibulous substrate, with the flow being, in sequence, from an addition site to the reaction site to a downstream site. In certain exemplary embodiments the sample is added to an addition site located centrally of one or more reaction sites located radially outward from the addition site. In multiplexed versions of such radial flow embodiments, the reaction site for one target analyte may be radially and/or circumferentially spaced from the reaction site for another target analyte. The nanocrystals are rendered detectably fluorescent by the aforesaid displacement of the quenching conjugate by the target analyte (if any) in the sample fluid. As noted above, displacement may be based, for example, on differential affinity and/or establishment of equilibrium between the

conjugates and the target analyte (if any) in the sample. It will be within the ability of those skilled in the art, given the advantage of this disclosure, to establish the displacement properties of the conjugates by the target analyte under assay conditions, whereby fluorescence can be correlated qualitatively to the presence or absence of the target analyte in the sample (or to the presence or absence of the target analyte above a threshold quantity) or quantitatively to the amount of target analyte in the sample. A pre-selected quantity of sample may be used (with or without buffering or other treatment) in certain exemplary qualitative or quantitative embodiments.

[16] In certain exemplary multiplexed embodiments of the lateral flow assays disclosed immediately above, a bibulous substrate of the assay component has multiple spatially separate (although optionally contiguous or not) reaction sites, each provided with nanocrystals quenched by conjugates bound to a layer of quantity of antibody or Fab fragments (as described immediately above) for a different target analyte. Thus, the binding sites and quenching conjugates at a first reaction site of the bibulous substrate is operative for determining a first target analyte. The binding sites and quenching conjugates at a second reaction site of the bibulous substrate is operative for determining a second target analyte, and so on. The fluorescence band optionally is different for the different reaction sites.

[17] The bibulous substrate may be formed of any suitable material, e.g., nitrocellulose. As with other embodiments of the assay systems disclosed here, the assay component of such lateral flow embodiments may be pre-loaded into a hand-held housing incorporating a fiuorometer and/or other components of the device, such as timing circuitry, communication ports for data output such as the assay results and in some embodiments for data input, a CPU such as a microprocessor, RAM or ROM or other

electronic memory, a user interface for the user to enter information about the assay or the sample source such as a graphical user interface, keyboard, touch screen etc., a display device for displaying assay results or other information, etc. It will be understood by those skilled in the art that at least certain exemplary embodiments of the lateral flow assays disclosed here, as for other embodiments, may be FRET-based systems.

[18] In certain exemplary embodiments, as discussed further below, the nanocrystals reagent comprise nanocrystals studded with immunotopic conjugates, either in the form of intact monoclonal antibodies (mAbs) or cleaved Fab fragments. Suitable mAbs and Fabs are commercially available or can be prepared in accordance with known techniques for many analytes of interest. For these and other embodiments of the disclosed assays, such immunotopic conjugates can be immobilized on the nanocrystal, for example, by conventional methods, such as by N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropylcarbodiimide (NHS/EDC) coupling chemistry. The antigenic quenching reagent is then prepared with saturating amounts of quenching conjugate, e.g., purified analyte-colloidal gold conjugates (CGC). Optionally the binding sites are specific for the target analyte.

[19] Certain other exemplary lateral flow embodiments of the assay systems disclosed here comprise an assay component incorporating one or more porous or absorbent or bibulous substrates (for convenience all referred to here as bibulous), e.g., in the shape or form of pads, strips or the like, wherein one or more of such bibulous substrates has a nanocrystal reagent (or multiple nanocrystal reagents for multiplexed embodiments), e.g., reversibly quenched fluorescent nanocrystals (referred to in some instances as "coated and quenched nanocrystal reagents") at one or more reaction

sites. The fluid sample is added to the bibulous substrate at a sample addition site. As noted above generally for the assay systems disclosed here, the reaction site may be at the same location as other sites of the assay component, such as the sample addition site, the detection site, etc. In certain exemplary embodiments, one or more quenching conjugates are associated with the nanocrystal to render it initially non- fluorescent under assay conditions. More specifically, the quenching conjugates comprise the target analyte (or a suitable analog or other substitute for the target analyte) conjugated to a quenching moiety, such as colloidal gold. The quenching conjugates are displaceably bound to antibodies or Fab fragments or other suitable affinity or binding sites (for convenience, as noted above, all referred to here generally as binding sites) for the target analyte. If the antibodies for a particular target analyte are so large that the colloidal gold or other quenching moiety is located too far from the nanocrystal to effectively or adequately quench its fluorescence, smaller sized Fab fragments may be employed. A polymer or other suitable coating on the nanocrystal carries the binding sites, e.g., by conjugating the antibody, Fab fragment, etc. Nanocrystals suitably coated to carry antibodies or Fab fragments in the lateral flow embodiments of the assay systems being discussed here are commercially available, e.g., EviTag® nanocrystal products available from Evident Technology (Troy, New York), or can be prepared using commercially known materials and techniques whose applicability will be recognized by those skilled in the art given the benefit of this disclosure. The coated nanocrystal with one or more antibodies or Fab fragments, which would otherwise fluoresce on exposure to excitation radiation, e.g., laser light of the appropriate wavelength provided by a fluorometer optionally provided as a component of the assay system, is quenched by the one or more conjugates bound to the nanocrystal at the binding site(s). In the

course of an assay, however, the quenching conjugate is displaced by the target analyte (if present in the sample fluid), generally in the manner discussed above, rendering the reaction site or detection site detectably fluorescent to indicate the presence of the target analyte in the sample fluid. The determination of target analyte may be either quantitative or qualitative, depending on the embodiment. In certain exemplary embodiments the quenching conjugate undergoes concentration dependent competitive displacement by analyte present in the sample.

[20] The coated and quenched nanocrystal reagent, i.e., the nanocrystals with antibody or other binding sites carrying quenching conjugates, may be incorporated into the bibulous substrate in any suitable manner, e.g., during manufacture of the bibulous substrate or by incorporation into the bibulous substrate after it is formed, etc. The coated and quenched nanocrystal reagent may be incorporated, e.g., by impregnation into or onto the bibulous substrate, etc. Upon exposure to the target analyte in a sample fluid, in certain exemplary embodiments some or all of the conjugates displaced by the target analyte are transported away from the nanocrystals by a flow of sample fluid. In certain exemplary embodiments the quenching conjugates are transported downstream from the reaction site along a generally linear bibulous substrate, with the flow being, in sequence, from an addition site to the reaction site to a downstream site. In certain exemplary embodiments the sample is added to an addition site located centrally of one or more reaction sites located radially outward from the addition site. In multiplexed versions of such radial flow embodiments, the reaction site for one target analyte may be radially and/or circumferentially spaced from the reaction site for another target analyte. As noted above, displacement may be based, for example, on differential affinity (of the target analyte vs. the quenching

conjugate for the binding site) and/or establishment of equilibrium between the conjugate and the target analyte (if any) in the sample. It will be within the ability of those skilled in the art, given the advantage of this disclosure, to establish the displacement properties of the conjugates by the target analyte under assay conditions, whereby the fluorescence can be correlated qualitatively to the presence or absence of the target analyte in the sample (or to the presence or absence of the target analyte above a threshold quantity) or quantitatively to the amount of target analyte in the sample. A pre-selected quantity of sample may be used (with or without buffering or other treatment) in certain exemplary qualitative or quantitative embodiments.

[21] In certain exemplary multiplexed embodiments of the lateral flow assays using coated and quenched nanocrystal reagents, a bibulous substrate of the assay component has multiple spatially separate (although optionally contiguous or not) reaction sites, each provided with nanocrystals quenched by conjugates bound to antibody or Fab fragments (as described immediately above) for a different target analyte. Thus, the binding sites and quenching conjugates at a first reaction site of the bibulous substrate are operative for determining a first target analyte. The binding sites and quenching conjugates at a second reaction site of the bibulous substrate are operative for determining a second target analyte, and so on. The fluorescence band optionally is different for the different reaction sites.

[22] As with other versions of the assay systems disclosed here, the assay component of such lateral flow assay systems may be loaded into a hand-held housing incorporating a fiuorometer and/or other components of the device, such as timing circuitry, communication ports for data output such as the assay results and in some embodiments for data input, a CPU such as a microprocessor, RAM or ROM or other

electronic memory, a user interface for the user to enter information about the assay or the sample source such as a graphical user interface, keyboard, touch screen etc., a display device for displaying assay results or other.

[23] Data input to (or received from) any of the assay devices or associated equipment and the like disclosed here can include, for example, any or all of the following: sample source information, time and date of assay, device serial number, assay performed, assay results, results, etc. Communication can be wired or wireless and can be conducted in any suitable format, e.g., ASCI format, etc.

[24] In accordance with one aspect, a fluorescence-based system, such as a device or method, is provided for determining the presence of at least one analyte in a sample, e.g., a chemical process by-product, contaminant, residue, biomolecule or moiety, etc. More specifically, the system comprises a single-use test site containing conjugated nanocrystal fluorophor (meaning a suitable quantity thereof) for a corresponding analyte, i.e., the target analyte. The target analyte of the assay may be either the actual molecule or moiety of interest or a derivative thereof resulting from modification, pre-conditioning or other treatment of the sample. The conjugated nanocrystal fluorophor has the property of being fluorescent in a predetermined wavelength range after being complexed with the corresponding analyte and exposed to suitable excitation light. In at least certain exemplary embodiments the fluorophor has a semiconductor nanocrystal core with an outer organic coating. The coating quenches the fluorescence of the nanocrystal core, that is, the coating prevents fluorescence by the nanocrystal so long as the coating remains intact. The fluorophor has one or more conjugates which are reactive or otherwise interactive with the target analyte under the assay conditions, e.g., in the aqueous or other solvent system of the

assay, in the pH range of the assay, etc. In certain exemplary embodiments each coated nanocrystal core carries one or more such conjugates, e.g., by attachment to the coating. The conjugates may in certain exemplary embodiments be referred to as analyte affinity conjugates or analyte affinity binding sites or the like. The coating on the nanocrystal has the property of being disrupted when an analyte affinity conjugate of the fluorophor complexes with the target analyte. That is, the coating is disrupted at least in the area of that particular complexed conjugate, such that the nanocrystal is at least partly unquenched and so rendered fluorescent under exposure to suitable excitation light, e.g., laser illumination.

[25] The fluorescence-based systems according to this aspect further provide an access port, e.g. a fluid injection or feed port, for combining fluid sample and conjugated nanocrystal fluorophor. For example, in certain exemplary embodiments the feed port may comprise a self-sealing injection port, a re-closable opening, a gravity feed tube, a wick, the outlet of a fluid line from a pump, or other fluid application device. Fluid sample (with or without treatment, additives, etc.) may, for example, be injected, pumped or otherwise fed into a reaction site of the assay system which already contains a quantity of the conjugated nanocrystal fluorophor in aqueous or other fluid system. Access is provided for fluid sample to the test site, such that conjugated nanocrystal fluorophor (again, as noted above, meaning a suitable quantity thereof) in the test site is exposed to the target analyte, if any, in the sample fluid.

[26] Certain embodiments of the assay provide a source of excitation light and observation access by which excitation light can enter the test site to expose the conjugated nanocrystal fluorophor and fluorescence can be observed from the conjugated nanocrystal fluorophores (if they are complexed with the corresponding analyte). As

noted above, the conjugated nanocrystal fluorophores are unquenched by such complexing, i.e., rendered fluorescent, when complexed with the analyte, e.g., by reaction or other interaction. The observation access may be, e.g., a window to the test site. It should be understood that the test site of the assay systems disclosed here may comprise multiple zones or portions, such that reaction between the conjugated nanocrystal fluorophor and the analyte occurs in one part of the test site and observation access is provided at another part of the test site to which the reaction products migrate or are pumped, fed by gravity or otherwise transported.

[27] Certain embodiments of the assay systems disclosed here also provide a light detector, e.g., a spectrometer or other suitable detector, operative to detect at least one wavelength of light in the wavelength range emitted by unquenched conjugated nanocrystal fluorophores, i.e., conjugated nanocrystal fluorophores complexed with the corresponding analyte, if any, in the sample, upon exposure to excitation light.

[28] In certain embodiments of the assay systems disclosed here, at least the test site, conjugated nanocrystal fluorophor and access for the fluid sample are integrated into a shelf-stable, hand-held test device. In certain such embodiments, the conjugated nanocrystal fluorophores are pre-packaged into the test site, e.g., in dry or dehydrated form or in an aqueous or other liquid, and the aforesaid access port is a feed port for fluid sample into the test site. Optionally, the observation access is also incorporated into such a hand-held test device. Optionally, a laser or other source of excitation light is also incorporated into such a hand-held test device. Optionally, the fluorescence detector is also incorporated into such a hand-held test device. Alternatively, the observation access is achieved by removing the test site from the hand-held test device and installing it (e.g., inserting, mounting or otherwise

connecting it) to a reading appliance which provides excitation light and a suitable detector for the resulting fluorescence, if any. In certain such embodiments the observation access may be automatically put into registration or alignment with the source of excitation light and the fluorescence detector by such installation.

[29] Optionally, in a multiplexed embodiment of the assay systems disclosed here, conjugated nanocrystal fluorophor for a second analyte is also provided at the test site, and optionally conjugated nanocrystal fluorophor for a third analyte, and so on. Preferably, each such conjugated nanocrystal fluorophor, if and when unquenched by interaction with a corresponding analyte, has a different fluorescence peak, such that its fluorescence can be distinguished from the fluorescence of the conjugated nanocrystal fluorophor for a different analyte. In accordance with such multiplexed embodiments, multiple analytes can be determined by a single system, simultaneously or sequentially.

[30] In accordance with another aspect, a fluorescence-based system, such as a device or method, is provided for determining the presence of at least one biomoleculear analyte in a biological fluid sample. More specifically, the system comprises a single- use test site containing conjugated nanocrystal fluorophor for a corresponding biomolecular analyte. The conjugated nanocrystal fluorophor, when unquenched by complexing with the corresponding biomolecular analyte and being exposed to suitable excitation light, is fluorescent in a predetermined wave length. In at least certain exemplary embodiments the fluorophor has an organic coating on a core semiconductor nanocrystal, and one or more conjugates, also referred to as analyte affinity conjugates or analyte affinity binding sites or bioaffinity conjugates or the like, which will complex with molecules or moieties of a target biomolecule analyte.

The coating quenches the fluorescence of the naiiocrystal core so long as it remains intact. The coating on the nanocrystal core has one or more conjugates, also referred to as analyte affinity conjugates or analyte affinity binding sites or the like, which will complex with molecules or moieties of a target biomolecular analyte. The target biomolecular analyte may be either the original biomolecule or a derivative thereof resulting from modification, pre-conditioning or other treatment of the original biomolecule In the biological fluid sample. Access is provided for a biological fluid sample to the test site, such that conjugated nanocrystal fiuorophor in the test site is exposed to the target analyte, if any, in the sample fluid.

[31] The assay system according to this aspect optionally also provides a source of excitation liφt as well as observation access including access for the excitation light to the test site and egress from the test site for fluorescence emitted by the conjugated nanocrystal fiuorophor. Thus, such observation access is a window, e.g., a transparent wall or other light path by which fluorescence can be observed from the conjugated nanocrystal fluorophores after they are complexed with the corresponding biomolecular analyte (if any) in the sample being tested.

[32] The assay system according to this aspect optionally also provides a detector for fluorescence emitted from the test site, generally in accordance with the discussion of such feature, above. Thus, in certain exemplary embodiments the system further comprises a light detector, e.g., a spectrometer, fluorometer or other suitable detector, operative to detect at least one wavelength of light in said predetermined wavelength range emitted by unquenched conjugated nanocrystal fluorophores, i.e., by conjugated nanocrystal fiuorophor complexed with the corresponding biomolecule analyte, if any, upon exposure to said excitation light. At least the test site and the access for the

biological fluid sample to the test site are integrated into a shelf-stable, storable, handheld test device.

[33] In certain exemplary embodiments of the fluorescence-based systems disclosed here for conducting an assay of a biomolecular analyte, the single-use test site containing conjugated nanocrystal fluorophor for one or more analytes and having access for combining the conjugated nanocrystal fluorophor with sample fluid is housed or packaged in a hand-held device, as described above.

[34] Optionally, in hand-held embodiments or versions of the assay systems disclosed here, the aforesaid source of excitation light and the sensor(s) (e.g., one or more spectrometers, etc.) for detecting or measuring fluorescence emitted from the test site, may be either on-board the hand-held device or provided in a separate appliance, e.g., an appliance having a receiving site adapted to receive and hold the hand-held device, e.g., a socket or a mounting port, hi certain exemplary embodiments such receiving site provides electronic signal communication to the hand-held device, e.g., to send and/or receive signals corresponding to the identity of the hand-held device (model, serial number, etc.), the nature, time, or date of the test performed, the source of the sample, quality control information, etc. Alternatively, such data communication can be conducted wirelessly from the hand-held device to the appliance. In embodiments not involving a separate appliance to read assay results, wireless communication from the hand-held device can be directly to any suitable communication port of a wireless LAN or other wired or wireless communication system. Any suitable present or future communication system, protocol or technique may be employed, such as a wireless LAN modem, Bluetooth, WI-Max, etc.

[35] In certain exemplary embodiments the observation access of the assay system is a window to the test site which registers or aligns with the source of excitation light and the fluorescence detector upon installation in the appliance.

[36] Optionally, in certain exemplary embodiments of the assay systems disclosed here, conjugated nanocrystal fluorophor for a second biomolecular analyte, e.g., for a second biomolecular analyte if the assay is to target biomolecular analytes, is also contained in the test site, and optionally conjugated nanocrystal fluorophor for a third biomolecular analyte, and so on. In certain exemplary embodiments conjugated nanocrystal fluorophor for each such analyte, if and when unquenched, by reaction with its corresponding analyte, has a different fluorescence peak such that its fluorescence can be distinguished from that of the conjugated nanocrystal fluorophor for a different one of the analytes being determined. In accordance with such multiplexed embodiments, multiple biomolecular analytes can be determined by a single system, optionally simultaneously or sequentially. Simultaneous determination can be performed, for example, if the different conjugated nanocrystal fluorophores are included in the same fluid system at the test site, or are provided in any other suitable way as a mixture or in close proximity to one another at the test site. Sequential determination can be conducted, for example, if the different conjugated nanocrystal fluorophores are presented in sequential zones of the test site or are staged to be available sequentially at the same zone, etc.

[37] In accordance with certain exemplary embodiments, wherein the fluorescence assay devices disclosed here are operative for assay of a single analyte, each of the conjugated nanocrystal fluorophores (i.e., each individual coated nanocrystal) carries one or more analyte affinity conjugates for the single analyte of interest. In

accordance with other exemplary embodiments, wherein the fluorescence assay devices are operative for simultaneous or sequential assay of multiple analytes., the conjugated nanocrystal fluorophores carry analyte affinity conjugates for each of the multiple analytes of interest. In certain such embodiments, optionally, the analyte affinity conjugates for each of the analytes are carried by a suitable quantity of conjugated nanocrystal fluorophores that do not carry analyte affinity conjugates for any other(s) of the multiple analytes. In alternative embodiments, individual coated nanocrystals may carry analyte affinity conjugates for multiple analytes, with the observation of corresponding fluorescence being interpreted accordingly as indicating the presence of one or more of those analytes. Subsequent assays may then be performed which distinguish amongst the multiple candidate analytes to arrive at a final definitive assay result.

[38] In accordance with certain exemplary embodiments operative to assay multiple analytes, either serially or simultaneously, a first type or species of analyte-specific conjugated nanocrystal fluorophor carrying analyte affinity conjugates only for a first analyte is provided at a test site of the assay device and analyte-specific conjugated nanocrystal fluorophor carrying analyte affinity conjugates only for a second analyte is provided at the same or a different zone of the test site in the device. The different zones of the test site are not all necessarily (always or at any time) in fluid communication with any or every other zone. Similarly, third, fourth, etc. species of analyte-specific conjugated nanocrystal fluorophor carrying analyte affinity conjugates for a third analyte, a fourth analyte, etc., respectively, may also be provided in the assay device, again, at the same or different zone(s) of the test site. In such multiplexed embodiments employing mono-analyte conjugated nanocrystal

fluorophores, detection of the various different target analytes can rely upon special and/or temporal separation of the fluorophores for one target analyte from the fluorophores for any or all other target analytes and/or on different emission wavelengths.

[39] Any or all of the target analytes may be pre-treated, as discussed above. If multiple different treatment reagents are employed, they optionally are presented at the same or different treatment zones.

[40] In certain exemplary embodiments the conjugated nanocrystal fluorophor for multiple, different analytes are immobilized on a substrate. Optionally, each different conjugated nanocrystal fluorophor is immobilized at a separate area of the substrate. The separate areas need only be sufficiently separate so as to be visually distinct from each other at or through the observation access. Thus, for example, the conjugated nanocrystal fluorophor for each of multiple analytes to be determined by the device or method can be arrayed at a corresponding one of a set of lines all radiating outwardly from a common center point. The lines instead can be arrayed parallel to one another. The conjugated nanocrystal fluorophores can alternatively be presented in a series of squares, circles or other shaped spots scored onto the substrate surface, spaced from each other or touching at their edges. Numerous alternative arrangements suitable for particular intended uses of the device, method or system will be apparent to those skilled in the art given the benefit of this disclosure.

[41] In certain multiplexed embodiments, especially, for example, those intended for use in a cascaded or multi-stage assay strategy, each of the nanocrystal fluorophores has analyte affinity conjugates for multiple different target analytes. Detection of

fluorescence in such multiplexed system indicates the presence of at least one of the target analytes, but does not necessarily indicate which of the analytes is (are) present. Optionally, further assays may then be performed, e.g., with mono-analyte assays, each specific to a single one of the target assays, such that the identity of the analytes present in the sample fluid is determined.

[42] Optionally, the single-use test site of the hand-held devices disclosed here is replaceable, such that a housing or other part(s) or component(s) of the hand-held assay device can be re-used. In certain exemplary embodiments replacement of the single-use test site comprises removal of a used fluid chamber or substrate having conjugated nanocrystal fluorophor(es) that have been exposed to a biological fluid sample, e.g., a quantity of saliva, and insertion or attachment or other loading of a fresh test site.

[43] In accordance with certain exemplary embodiments, the quantum dots reagent, i.e., the_conjugated nanocrystal fluorophores, optionally referred to as conjugated quantum dots, are provided in solution, e.g., an aqueous solution, in a suitable reaction vessel or are otherwise presented at the test site of the hand-held assay device for exposure to a biological fluid sample, e.g., diluted or undiluted saliva, urine, serum, etc. In other exemplary embodiments conjugated nanocrystal fluorophores are provided in a dry state, immobilized or mobilizable on a substrate, e.g., on an impregnated fibrous layer (meaning in or on the fibers, etc.), a plate, etc. Other suitable techniques for presenting the conjugated nanocrystal fluorophores at the test site of the hand-held device or otherwise providing them in assay systems in accordance with the present invention will be apparent to those skilled in the art given the benefit of this disclosure.

[44] In certain exemplary embodiments of the assay systems disclosed here, the conjugated nanocrystal fluorophor(es) (i.e., each type of such fluorophor or each fiuorophor corresponding to a particular analyte) is analyte-specific. The fluorophor will complex with that one particular analyte and not with any other molecules or moieties expected or likely to be in the sample fluid under the test conditions described. More specifically, as used here, an analyte affinity conjugate is "specific" or "analyte specific" for a corresponding analyte if it is reactive only with that analyte or if it is reactive with the corresponding analyte and either non-reactive or not significantly reactive under the assay conditions with any other molecules or moieties expected to be present in the sample fluid. An analyte affinity conjugate is not significantly reactive with such other molecules or moieties if its reactivity with such other molecules or moieties does not adversely impact the accuracy of the assay at a measurable level or at a level which unacceptably diminishes or otherwise degrades the accuracy of the assay to below acceptable levels. The number of molecules or moieties of an analyte that can bind to or complex with a single conjugated nanocrystal fluorophor will typically depend on the number of binding sites carried by that fluorophor, i.e., conjugated to the coating, steric hindrance, etc. The analyte specific conjugated nanocrystal fluorophor will typically have multiple conjugates available to complex with the corresponding analyte. Moreover, the fluorophores are analyte-specific also in that they will not complex (under the conditions of the test) with any other analyte likely to be in the biological sample in a manner effective to unquench and hence render fluorescent the fluorophores.

[45] In accordance with another aspect, a fluorescence-based system is provided for determining the presence of any of multiple analytes, e.g., multiple biomolecular

analytes in a biological fluid sample. The system comprises a single-use test site containing at least one multiplexed conjugated nanocrystal fluorophor, i.e., a suitable quantity of at least a first multiplexed conjugated nanocrystal fluorophor and optionally a suitable quantity of a second conjugated nanocrystal fluorophor, a third, etc. Such conjugated nanocrystal fluorophor, if any, optionally also is a multiplexed conjugated nanocrystal fluorophor, a third, etc. The conjugated nanocrystal fluorophor is "multiplexed" in that it has the property of being non-fluorescent unless complexed with any one or more of multiple different analytes and then exposed to suitable excitation light. Thus the multiplexed conjugated nanocrystal fluorophor of an assay system according to this aspect of the disclosure is non-analyte-specific, meaning that coated nanocrystal has analyte affinity conjugates which will complex with any of multiple corresponding analytes in a manner effective to unquench the fluorophor, i.e., render it fluorescent under suitable excitation light. In accordance with certain exemplary embodiments the multiplexed fluorophor has one or more conjugates which are analyte specific for a first biomolecular analyte, and also has other conjugates which are analyte specific for a second, different biomolecular analyte, and possible other conjugates for a third, fourth and so on. In accordance with certain exemplary embodiments the multiplexed fluorophor has one or more conjugates all of which are analyte-specific for a multiple biomolecular analytes. Certain exemplary embodiments combine fluorophores of these different types, hi all such embodiment(s), preferably, the multiplexed fluorophor will not complex (under the conditions of the test) with any other analyte(s) likely to be in the biological sample in a manner effective to unquench the fluorophor and hence render it spuriously fluorescent. Thus, in accordance with this aspect, it can be determined that at least one of the analytes corresponding to a fluorescing fluorophor is present in the

biological sample, although which of such analytes may not be determinable from this one assay. Fluorescence-based systems in accordance with this aspect otherwise are similar to those disclosed above. In accordance with certain exemplary embodiments, multiple multiplexed fluorophores are used in the test site. Preferably, each of the multiplexed fluorophores is specific for a different, non-overlapping set of biomolecular analytes, and preferably each has a different fluorescence peak when unquenched. Thus, detecting a particular fluorescence peak indicates which fluorophor was unquenched, and therefore, indicates that at least one of the analytes to which that fluorophor corresponds was present in the fluid sample.

[46] Optionally, multiplexed methods and devices can be configured for use in accordance with this disclosure in a tiered manner. That is, additional assays can be performed to determine which one(s) of the corresponding analytes is present when the fluorescence peak of a multiplexed conjugated nanocrystal fluorophor is observed. Thus, in certain exemplary embodiments of such systems, an assay is conducted using one or more multiplexed conjugated nanocrystal fluorophores in the test site of a first hand-held device. If a hit occurs, i.e., the fluorophor is unquenched, one or more second tier devices can then be employed having single-analyte conjugated nanocrystal fluorophores to determine which analyte or analytes are present in the sample. The second tier device preferably has a set of analyte-specific conjugated nanocrystal fluorophores, specifically, a quantity of conjugated nanocrystal fluorophor which is analyte-specific for a first one of the analytes multiplexed in the fluorophor unquenched in the first assay, a quantity of conjugated nanocrystal fluorophor which is analyte-specific for a second one of the analytes multiplexed in the fluorophor unquenched in the first assay, and so on. In this manner, a fluid

sample can be quickly, effectively and economically assayed. Other such strategies for employing the fluorescence-based systems disclosed here in a tiered manner will be apparent to those skilled in the art given the benefit of this disclosure.

[47] The analyte(s) determined by any particular embodiment of the assay systems disclosed here may be any analytes for which suitable conjugated nanocrystal fluorophor can be prepared. In the case of assays for the determination of biomolecular analytes, the biomolecular analyte(s) of interest may be any potentially present in a biological fluid sample to be tested (or a derivative of such biomolecular entity), including, for example, a drug-of-abuse, e.g., an opiate, THC, an amphetamine, etc. or a metabolite of such a drug, an induced or naturally occurring disease marker, or an administered or prescribed drug. An administered or prescribed drug may be assayed, for example, to confirm that a patient is taking (or that she/he is properly taking) the drug or that correct serum levels are being reached or maintained. A drug-of-abuse may be assayed, for example, to qualify a new job candidate or to determine adherence to a drug rehabilitation program. A disease marker may be assayed, for example, as an aid to diagnosis, treatment or prevention of disease. Other biomolecules for which the fluorescence-based systems of this disclosure may be employed will be apparent to those skilled in the art given the benefit of this disclosure.

[48] An analyte affinity conjugate for a target analyte, suitable for at least certain exemplary embodiments of the assays disclosed here, may comprise any suitable molecule or moiety which can be conjugated to the nanocrystal fluorophor and which is capable of reaction or other suitable interaction(s) with the analyte. In the case of biomolecular analytes, for example, the analyte affinity conjugate comprises any

suitable molecule or moiety which can be conjugated to the coated nanocrystal fluorophor and which is capable of suitable interaction(s) of biological affinity or other suitable reaction, e.g., a binding reaction, with the target analyte, i.e., with the original biomolecular analyte in the biological sample fluid or with a naturally occurring or treatment-generated derivative thereof. Exemplary analyte affinity conjugates include antibodies, aptamers and other oligomers, and other suitable entities capable of being conjugated to the nanocrystal fluorophor, e.g., to a polymer coating on the semiconductor core of the conjugated nanocrystal fluorophor, and capable in that condition of taking advantage of the target analyte's bioaffmity binding properties, such as, for example, by reaction with any of the analyte's one or more binding sites. In accordance with certain exemplary embodiments, the use of antibody-based conjugates provides significant improvement in assay performance over products and processes previously available. Teachings regarding aptamers are provided, for example, in the following citations which those skilled in the art will be able to apply to the assay systems disclosed here, given the benefit of this disclosure: Jayasena SD (1999) "Aptamers: An Emerging Class of Molecules that rival antibodies in diagnostics," Clin. Client. 45:1628-1650; Stojanovic MN, de Prada P, Landry DW (2000) "Fluorescent sensors based on aptamer self-assembly," J. Am. Chem. Soc. 122:11547-11548; Stojanovic MN, de Prada P, Landry DW (2001) "Aptamer-based folding fluorescent sensor for cocaine," J. Am. Chem. Soc. 123:4928- 4931; and Stojanovic MN, Landry DW (2002) "Aptamer-based colorimetric probe for cocaine," J. Am. Chem. Soc. 124:9678-9679. In accordance with certain exemplary embodiments, the use of aptamer based conjugates advantageously provides sensitivities in the subnanomolar range. In certain preferred embodiments the increased selectivity of these synthetic polynucleotides will be found to provide

nearly a log unit improvement of measurable signal. Currently, signal this low can typically only be detected by sophisticated gas or liquid chromatography techniques coupled with single or tandem mass spectrometry. In accordance with other exemplary embodiments, the analyte affinity conjugates of the conjugated nanocrystal fluorophores may be receptor fragments, other macromolecules, etc. Other analyte affinity conjugates suitable for some or all of the fluorescence-based systems of this disclosure, depending in part on the intended use of the system, e.g., on the target analyte, the type of biological fluid to be tested, etc. will be apparent to those skilled in the art given the benefit of this disclosure.

[49] Optionally, other nanocrystal fluorophores, referred to here in some cases as control fluorophores or, if conjugated, optionally referred to as control/calibrator conjugated nanocrystal fluorophores or control/calibrator conjugated fluorophores, can be provided at the treatment site (if any), test site on at a control site of the assay device. In certain exemplary embodiments such control/calibrator conjugated nanocrystal fluorophores carry conjugates reactive with a control/calibrator agent. For example, in certain exemplary embodiments such control/calibrator conjugated nanocrystal fluorophores can serve to confirm that the assay device is properly operative at the time of use, e.g., to validate instrumentation/methods between sites/analyzers/laboratories, or to act as proficiency tests, etc.

[50] In accordance with certain exemplary embodiments of the fluorescence-based systems disclosed here for determining the presence of analytes in a fluid sample, the methods, devices, etc. are suitable for use in forensic assays, more specifically, detection of substances where there are medical-legal concerns, i.e., poisons (arsenic, etc), pharmaceuticals (GHB, rohypnol, etc) illicit drugs (ecstasy, methamphetamine, etc.).

In other exemplary embodiments of the fluorescence-based systems disclosed here for determining the presence of analytes in a fluid sample, the methods, devices, etc. are suitable for use in clinical diagnostic assays, more specifically, the identification of specific viruses, bacteria or viral/bacterial subtypes, pharmaceuticals, cell markers, biomarkers (PSA, etc.) therapeutic drug monitoring, (digitalis, immunosuppressants) etc. In other exemplary embodiments of the fluorescence-based systems disclosed here for determining the presence of analytes in a biological fluid sample, the methods, devices, etc. are suitable for use in environmental assays, more specifically, identification of environmental toxins, industrial pollutants/byproduct/intermediates, toxic microorganisms, etc.

[51] hi accordance with another aspect, a fluorescence-based competitive immunoassay system is provided for determining the presence of at least one biomolecular analyte in a biological fluid sample. More specifically, the system comprises a single-use test site containing at least (i) conjugated nanocrystal fluorophor for a corresponding biomolecular analyte, and (ii) a competitor reactant, i.e., a reagent or reactant that will bind with the conjugated nanocrystal fluorophor in place of the analyte. hi certain exemplary embodiments the analyte can displace the competitor reagent from the conjugated nanocrystal fluorophor and thereby unquench the conjugated nanocrystal fluorophor. In such embodiments the presence of fluorescence indicates the presence of the analyte in the sample, hi certain exemplary embodiments the competitive reagent is able to react with the conjugated nanocrystal fluorophor only if the analyte is not present in the sample. In such embodiments the location of fluorescence, for example, indicates the presence or absence of the analyte, e.g., if the competitive reagent is bound or fixed to a certain location in the test site. Assay systems

according to this aspect follow generally the features and principles of operation set forth above.

[52] It will be appreciated by those skilled in the art, that is, by those having skill and experience in the technology areas involved in the novel fluorescence-based systems disclosed here for assaying biomolecular analytes, that significant advantages can be achieved by such systems. For example, in certain multiplexed embodiments described further below, multiple analytes can be determined, optionally simultaneously, using one hand-held device and a single source of excitation light to illuminate all of the multiple different types of conjugated nanocrystal fluorophores, i.e., the different conjugated nanocrystal fluorophores corresponding to the different analytes being determined. These and at least certain other embodiments of the systems, e.g., methods, devices etc., disclosed here are suitable to provide advantageous convenience, economy, accuracy and/or speed of testing. Additional advantages will be apparent to those skilled in the art given the benefit of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[53] Fig. 1 is a schematic diagram of a hand-held device for point-of-care assay of biomolecular analytes, in accordance with one exemplary embodiment of the present invention.

[54] Fig. 2 is an enlarged schematic diagram of a replaceable, single-use assay component suitable for use in the hand-held assay device of Fig. 1.

[55] Fig. 3 is an enlarged schematic diagram of a micro-bead suitable for use (in quantity) in the assay component of Fig. 2, comprising a plastic or polymer micro-bead

impregnated with intrinsically fluorescent nanocrystals and studded with quenching conjugates, that is, having a surface coating of quenching conjugates comprising antibodies for the target analyte displaceably bound to analyte/colloidal gold conjugates.

[56] Fig. 4 is a schematic diagram of a hand-held device for assay of multiple analytes in accordance with another exemplary embodiment of the present invention.

[57] Fig. 5 is an enlarged schematic diagram of a replaceable, single-use assay component suitable for use in the hand-held assay device of Fig. 4.

[58] Fig 6 is a schematic diagram of one exemplary embodiment of the assay component of Fig. 5

[59] Fig 7 is a schematic diagram of a second exemplary embodiment of the assay component of Fig. 5.

[60] The figures referred to above are not drawn necessarily to scale and should be understood to provide a representation of certain exemplary embodiments of the invention, illustrative of the principles involved. Some features depicted in the drawings have been enlarged or distorted relative to others to facilitate explanation and understanding. In some cases the same reference numbers may be used in drawings for similar or identical components and features shown in various alternative embodiments. Particular configurations, dimensions, orientations and the like for any particular embodiment will typically be determined, at least in part, by the intended application and by the environment in which it intended to be used.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

[61] The use of a semiconductor nanocrystals or "quantum dots" as a component of the signal or reporter construct in systems disclosed here provides significant improvement for several assay parameters. Such nanocrystals are excited by a single wavelength, typically by any one or more wavelengths within a wide wavelength range. Due to their inherent structural characteristics such nanocrystals emit at very defined wavelength peaks in the visible or infrared spectra. This property makes the measurement of multiple analytes in a single reaction chamber possible. Advantageously, multiple different conjugated nanocrystal fiuorophores in the same single-use test site of a device, each having a different emission peak, is excited by the same excitation light source. The emission spectra are very energy efficient, producing peaks that are extremely narrow and do not exhibit large tailing or shouldering. Such nanocrystals are readily conjugated to a suitable binding molecule or moiety for any of innumerable target analytes, e.g., aptamers, antibodies, receptor fragments, binding fragments, the binding domain of a receptor, Fabs, etc. Other suitable conjugates will be apparent to those skilled in the art given the benefit of this disclosure. The surface of nanocrystals can be modified, making possible various fluorescent signal "quenching" approaches. Nanocrystals suitable for the systems disclosed here for determination of an analyte in a fluid sample, e.g., for the determination of one or more different biomoleculular analytes in a biological fluid sample can be inexpensively and accurately produced in large quantities. Such nanocrystals are chemically stable, thus enhancing the "shelf life" of the disclosed systems. Few such nanocrystals are necessary to produce a measurable signal, thus reducing any potential toxicity from the assay itself.

[62] For purposes of convenience, the discussion below will focus primarily on certain exemplary embodiments of the assay systems disclosed here, wherein the assay systems are operative for determination of one or more biomolecular analytes in a biological fluid sample. It should be, understood that the principles of operation, system details, optional and alternative features, etc. are generally applicable as well to embodiments of the assay systems disclosed here that are operative for determination of one or more non-biomolecular analytes, e.g., chemical process products or by-products, contaminants, residues, toxins, reactants or reagents, etc.

[63] In the fluorescence-based systems discussed here for determining one or more analytes in a fluid sample, as noted above, at least the test site and the feed port or other access for the fluid sample to the test site are integrated into a shelf-stable, storable, hand-held test device. In certain exemplary embodiments the test site, access for the fluid sample, source of excitation light and fluorescence detector and optionally other components and features are integrated into the hand-held device, providing a stand-alone assay device, i.e., a device that is operative to perform the assay without involvement of other equipment once the fluid sample is introduced to the test site of the device. As used here and in the appended claims, a "hand-held" test device of the present disclosure means any device that is suitable to be held and operated by one person, e.g., by a process control, quality assurance or quality control technician, nurse, physician, lab technician etc. while introducing the biological or other fluid sample via the sample access to the test site. It should be understood in this regard that a device is a hand-held device as that term is used here and in the appended claims if it meets such criterion, even if it optionally can be used with a cradle, fixturing device or the like. As used here and in the appended claims, the

hand-held test devices of the present disclosure are "shelf-stable" if they can remain in storage at room temperature or at reduced temperature for a period of at least days without significant loss of accuracy, i.e., without losing accuracy to the extent that the device is unacceptable for its intended purpose. In accordance with certain exemplary embodiments, hand-held test devices are shelf-stable for at least months e.g., they remain suitably operative for at least 2 months, in some cases at least 4 months and in some cases at least 6 months or more. As used here and in the appended claims, the test site containing conjugated nanocrystal fiuorophores for at least one corresponding analyte is a "single-use" test site if it is not suitable in ordinary usage to be re-charged or re-loaded with new conjugated nanocrystal fiuorophores for the same or different analyte(s). That is, for example, the test site is a single-use test site if evacuation and re-charge would require permanent deformation or other damage to the test site or other component of the hand-held device. Thus, such test sites are single-use if there is no port or other access means designed for removal of the original conjugated nanocrystal fiuorophores and introduction of a charge of new conjugated nanocrystal fiuorophores. hi certain exemplary embodiments wherein the conjugated nanocrystal fiuorophores are provided in aqueous solution in the test site, for example, the test site is single-use if it does not have a fluid-tight evacuation and re-fill port.

[64] The target analyte may be the actual biomolecule originally in a biological fluid sample or a modification or other derivative thereof, i.e., the product of such original biomolecule and a reactant or other reagent, referred to here as a treatment reagent or pre-treatment reagent. Exemplary treatment reagents include diabodies having a first affinity or binding site for the target analyte and a second site for the conjugate of the conjugated nanocrystal fluorophor. In certain exemplary embodiments the target

analyte may be the product of a multi-step pre-treatment of the original biomolecule. Such treatment reagents may be provided at the same location or zone of the test site as the conjugated nano crystal fluorophor or, optionally, at a different zone of the test site. Treatment reagents preferably are provided at the test site (optionally referred to as the reaction site) of the hand-held device or at a treatment zone of the device. Various alternative treatment reagents will be apparent to those skilled in the art given the benefit of this disclosure and the needs of a particular application of the system.

[65] In certain exemplary embodiments of the fluorescence based systems disclosed here for determining the presence of one or more biomolecular analytes in a biological fluid sample, the test site is not in fluid communication with the biological fluid sample access in at least one condition and is operative in at least one operator- selectable condition to be in fluid communication with the biological fluid sample access. For example, in the embodiment illustrated in Figs. 1-3, a hand-held device 10 is seen to comprise a housing 12 containing a reaction site 14 having both a treatment zone 16 and a reaction zone 18. Device 10 is operative to determine multiple biomolecular analytes, i.e., to quantitatively measure each of multiple biomolecular analytes. Treatment zone 16 is a fluid tight chamber and reaction zone 18 is a second fluid-tight chamber. Optionally, the two chambers can be provided merely as portions of one common vial or container, with any suitable divider between them. Treatment zone 16 contains multiple diabodies in aqueous solution, i.e., a sufficient quantity of diabodies for each of the biomolecules to be determined by the assay. Thus, each of the biomolecular analytes (if present in the sample) is subjected to pre-treatment in the treatment zone before being passed to the test zone. The diabodies have a first affinity domain for the corresponding biomolecule and a

second, conditional affinity domain for the corresponding conjugates of the conjugated nanocrystal fluorophores. The second domain is a conditional affinity domain in that it is not operative to bind to the corresponding conjugated nanocrystal fluorophor under the assay conditions unless the first domain of that diabody is bound to the corresponding biomolecule. The reaction zone 18 contains conjugated nanocrystal fluorophores in aqueous solution for each of the multiple biomolecular analytes. More specifically, in the illustrated embodiment the conjugated nanocrystal fluorophores are provided all in one common aqueous solution. In this embodiment the assay performed by the test device is a multiplexed assay comprising multiple different types or species of conjugated nanocrystal fluorophores, wherein each such conjugated nanocrystal fluorophor has conjugates for only one of the target biomolecular analytes.

[66] In the embodiment of Figs. 1 - 3 the hand-held device optionally is pre-loaded in the shelf-storage condition, i.e., the condition in which the hand-held device is intended to be stored before use. That is, the device is shelf-stable with the assay component in place.

[67] The assay system 10 is seemed to comprise the reusable hand-held housing 12 and single-use, replaceable assay component 14. Assay component 14 is sized to be received into sample chamber 16 of housing 12. A fluorometer is housed within housing 12 and comprises a laser source of excitation light or illuminating the nanocrystal reagent of assay component 14 during the assay, as well as the sensor for detecting the characteristic fluorescents emitted by nanocrystal reagent as if the target analyte is present in the sample fluid. Housing 12 is seemed to further comprise a screen 18 operative to display information concerning the assay, including assay

results, etc. Housing 12 further comprises key pad 20 operative to receive information from an assay operator, as well as data port 22 operative communicate data, for example in ASCI file format or other suitable format. Sample fluid 24 is added to assay component 14 via access port 26. Sample fluid can be provided by any suitable means, for example, pipette, eye dropper, automatic or manual pump, etc. As best seen in Fig. 1, the illustrated embodiment employs a hand held sample collection and delivery device 25. In the illustrated embodiment, access 26 comprises a porous membrane 28. Alternatively, access for sample fluid to assay component 14 may comprise, for example, a wicking member, a capillary tube, etc. Conjugated nanocrystal fluorophores that complex with their corresponding analyte at the test or reaction site of assay component 14 are thereby unquenched and so rendered detectively fluorescent when exposed to excitation light by the fluorometer housed within housing 12. Assay component 14 is seen to comprise in addition to access 26, reaction site 30 comprising antibody-coded nanocrystal-impregnated beads, e.g. styrene beads, etc., as discussed above. Between access site 30 and access 26, in the line of flow of fluid sample added to assay component 14 is buffer reagent 32 for pre- treatment of sample fluid. In addition to buffer, those skilled in the art will recognize that additional reagents, diluance and other assay materials, such as a filter, etc. may be incorporated in the buffer region or the access region.

[68] Referring now to Fig. 3, a nanocrystal-impregnated bead is schematically illustrated. Specifically, microbead 36 is seen to incorporate fluorescent nanocrystals 38, at least some of which are at or near the surface of the bead. Antibodies or Fab fragments 40 that provide binding sites for the target analyte are associated with the bead, specifically, at the outer surface. Such binding sites may be referred to as a film or a

layer on the outer surface of the bead, however, it should be understood that such film or layer is not necessarily a continuous film and, rather, may be in the nature of individual antibodies or Fab fragments studded to the surface of the bead. Conjugates 42 of the target analyte and colloidal gold quenching moiety are bound to the antibodies or Fab fragments 40. The conjugates render the beads (that is, the nanocrystals thereof) substantially non-fluorescent when exposed to the excitation light under assay conditions. Upon exposure to target analyte in a sample fluid, however, some or all of the conjugates are displaced, thus rendering the beads detectably fluorescent. Detection of fluorescence, therefore, is indicative of the presence of target analyte in the sample fluid.

[69] Referring now to Fig. 4, an alternative embodiment of the assay systems disclosed here is seen to comprise a housing 12 substantially as described in connection with the embodiment of Figs. 1 - 3. The embodiment of Fig. 4 is a lateral flow embodiment employing assay component 48 comprising a bibulous strip 50. Sample fluid 24 is added to the bibulous strip at application zone 51 and is transported by adsorption or the like through the strip to multiple reaction zones 52 — 55. The embodiment of Fig. 4 is a multiplexed assay embodiment wherein each of the reaction sites 52 - 55 is configured for determination of a different target analyte. Assay component 48 is received into sample chamber 16 of hand-held housing 12 after application of sample fluid for determination of the presence of the various target analytes. Accordingly, the fluorometer incorporated in housing 12 is configured to determine some of the presence of fluorescence from each of reaction sites 52 - 55 separately. Such separate determination may be either sequential or simultaneous. The different reaction zones can be distinguished from each other by the fluorometer during the assay by any

suitable technique, including, based on each of the reaction zones 52 - 55 employing a nanocrystal reagent having a different characteristic fluorescence wavelength band. In addition or alternatively, the fluorometer may be configured to distinguish the reaction zones spaciously from one another. Also, the determination of fluorescence at each of the reaction zones may be chronologically separated. Additional techniques may be apparent to those skilled in the art given the benefit of this disclosure. Also, it will be recognized that one or more of the reaction sites may be employed as a control to confirm proper functioning of the assay. The area of the bibulous strip to the right of the reaction zones (as viewed in Figs. 4 — 6 may function as a "sink" or reservoir to receive sample fluid flowing from access 51 to and through the reaction zones during the assay. Figure 6 schematically illustrates the configuration of the reaction zones 52 - 55 in assay component 48. The nanocrystals are incorporated into the bibulous strip at the reaction zones, in the illustrated embodiment, so as to not be mobilizable or transportable by the flow of sample fluid during the assay. Nanocrystals 62 having a first characteristic wavelength are incorporated at reaction zone 52. Similarly, nanocrystals 63 having a characteristic wavelength different from that of nanocrystals 62 are incorporated at reaction zone 53. Nanocrystal 64 are embedded at reaction zone 54 and have a fluorescence wavelength different from that of nanocrystals 61 and 62. Finally, nanocrystals 65 are embedded at reaction zone 55 and have yet a different characteristic fluorescence wavelength. The fluorescence of nanocrystals 62 - 65 are quenched by their respective quenching conjugates. More particularly, at reaction zone 52 a layer or coating of a first anti-body 72 corresponding to a first target analyte is provided over the embedded nanocrystal 62. A conjugate 82 of the target analyte and the quenching moiety as colloidal gold is bound to the analyte layer 72 at reaction zone 52. In such

configuration, the colloidal gold quenching moieties quench fluorescence of the underlying nano crystals at reaction zone 52. If the antibodies 72 are sufficiently large so as to separate and render less effective the quenching moieties, smaller sized Fab fragments or the like can be employed. The antibody and analyte/quenching conjugates are bound to the analyte at the reaction zone are not mobilizable or transportable by a flow of sample fluid. If target analyte is present in the sample fluid, however, it displaces some or all of the analyte/quenching moiety conjugates, as discussed above, and such conjugates are transported by the flow of sample fluid out of reaction zone 52. The underlying nanocrystals 62 are thereby rendered fluorescent (i.e., unquenched) and detection of such fluorescent indicates the presence of target analyte in the sample.

[70] Similarly, antibodies 73 for a second target analyte are provided at reaction zone 53. In conjugate 83, the quenching moiety and such second analyte is bound to the analyte sub 3, thereby quenching nanocrystal 63 at reaction zone 53. In the manner described for reaction zone 52, the presence of such second target analyte in the sample fluid will cause conjugates 83 to be displaced from antibody 73 and transported by the flow of sample fluid (or other developing fluid) our of reaction zone 53 thereby rendering nanocrystals 63 detectably fluorescent by the fluorometer incorporating in housing 12. A third analyte can be detected at zone 54 using third antibodies 74 and corresponding conjugates 84. Similarly, antibody 75 at zone 55 carry conjugates 85 for determination of a fourth analyte. It will be apparent to those skilled in the art that alternative special arrangements of the reaction zones is possible, for example, radial, linear, etc. and that more or fewer reaction zones can be provided, depending upon intended use of the assay system.

[71] An alternatively embodiment of an assay component for use in a lateral flow assay is illustrated in Fig. 8. Assay component 90 comprises bibulous strip 92 arranged much like assay component 48 in Fig. 5. Sample fluid access is provided at zone 94 and sample fluid travels from their through the bibulous strip to reaction sites 95 - 98. Antibodies 101 for a first target analyte are carried on the surface of coated nanocrystals 102 having a first intrinsic fluorescence. Such fluorescence is quenched by analyte/quenching moiety conjugates 103 displaceably bound to antibodies 101. In the presence of target analyte in the sample fluid, some or all of conjugates 103 are displaced by such target analyte, thus rendering nanocrystals 102 detectably fluorescent. Assay component 90 can be inserted into a hand-held device the same as or similar to device 12 in Fig. 1 for detecting such fluorescence. Similarly, antibodies 104 for a second target analyte are bound to a second quantity of nanocrystals 105 incorporated into a bibulous strip at reaction zone 96. Quenching conjugates 106 quench the fluorescence of nanocrystals 105 unless displaced by the presence of the second target analyte in the sample fluid. Nanocrystals conjugated to antibodies or Fab fragments are commercially available or can be prepared using known techniques, whose applicability will be apparent to those skilled in the art given the benefit of this disclosure. Suitable nanocrystals conjugated to antibodies are commercially available, for example, from EviTags® from Evident Technology (Troy, New York). In similar fashion, quencher conjugates are presented at reaction zones 97 and 98 for third and fourth analytes, respectively.

[72] In accordance with certain exemplary embodiments, the single-use test site containing conjugated nanocrystal fluorophores, biological fluid sample access to the test site, source of excitation light and observation access by which fluorescence can be

observed from conjugated nanocrystal fluorophores complexed with the corresponding biomolecular analyte all are integrated into the hand-held device. In accordance with certain embodiments, the single-use test site, biological fluid sample access to the test site, source of excitation light, observation access, and source of excitation light all are integrated into the hand-held device. In other embodiments the source of excitation light and the fluorescence reader are provided in a reusable apparatus, e.g., desktop apparatus or the like having a receptacle suitable to receive and hold the test site device during the fluorescence reading step of the assay. Such apparatus optionally is in communication, e.g., via phone line, internet connection or the like, with central data acquisition, processing, storage and/or dissemination facilities as discussed further below.

[73] In accordance with certain exemplary embodiments of the systems disclosed here, the conjugated nanocrystal fluorophores and/or any treatment reagent can be provided on a solid support, such as, for example, the aforementioned bibulous substrate, such support being either soluble or insoluble depending on the delivery and assay technique to be employed by the system. Such solid support can be any suitable material, e.g., materials that provide a rigid or semi-rigid surface, or can be a fibrous or absorbent material. Exemplary solid supports include but are not limited-to pellets, fibrous mats such as a glass fiber mat, polyester fiber mat, etc, nitrocellous, bibulous material, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N'-bis-

acryloylethylenediamine, and glass particles. Optionally, the surface of the solid support may be treated, e.g., coated with a hydrophobic polymer, etc.

[74] As noted above, the access for a biological fluid sample to the test site of the fluorescence-based systems disclosed here for determining at least one biomolecular analyte in the biological fluid sample may be provided in any of numerous alternative forms. Typically the access means will depend, in part, on the form in which the conjugated nanocrystal fluorophores and any treatment reagent are provided. In certain exemplary embodiments the access, i.e., the pathway, channel or delivery mechanism for sample fluid to the test site, comprises a wicking member. For example, sample fluid can be applied to a sample application zone of a wicking member and delivered by wicking action to the test site. Optionally the test site is a downstream portion or zone of the same wicking member or is a site (e.g., an adjacent absorbent member) with which the wicking member is in fluid communication. In certain such embodiments the conjugated nanocrystal fluorophores and/or any treatment reagent may be provided by immobilizing them on the downstream zone of the wicking member which will serve as the test site. The sample fluid can be applied to the wicking member in any suitable manner, e.g., by eye dropper, pipette, etc. Where the conjugated nanocrystal fluorophores and/or treatment agent, if any, is provided in solution, the access for a biological fluid sample to the test site may be provided, for example, by plenum for needle injection, openable port, e.g., a capillary tube or the like having an openable gate or the like, or by other suitable means. Numerous other suitable access devices and techniques will be apparent to those skilled in the art given the benefit of this disclosure.

[75] Consistent with the discussion above, in certain exemplary embodiments of the fluorescence-based systems disclosed here for determining the presence of at least one biomolecular analyte in a biological fluid sample, the biological fluid sample access to the test site involves fluid communicatien first to a treatment zone, with the treatment zone and the reaction zone being not in fluid communication with one another (i.e., being in fluid isolation from one another) in a first condition while the biological fluid sample is first introduced, and then subsequently in fluid communication with one another in an operator-selectable second condition.

[76] In certain exemplary embodiments of the fluorescence-based systems disclosed here, the hand-held test device is suitable to receive a patient's biological fluid sample at a patient point-of-care location.

[77] It should be understood that reference here and in the appended claims to a conjugated nanocrystal fiuorophor, unless otherwise clear from context, means a quantity sufficient in the device or system to determine (i.e., detect and/or quantify, in accordance with the particular intended use of the system) the biomolecular analyte of interest, i.e., the target analyte. The quantity of conjugated nanocrystal fluorophor should all be of suitably consistent properties. Such properties include, at least, biological affinity for the biomolecular analyte target and optical properties when quenched (i.e., prior to being complexed with the analyte) and when unquenched and exposed to suitable laser or other excitation illumination. In this regard, it is acceptable in at least certain embodiments that a quantity of non-conforming conjugated nanocrystal fluorophores also be present, e.g., "out-of-spec" conjugated nanocrystal fluorophores, so long as the performance of the "in spec" conjugated nanocrystal fluorophor for the biomolecular analyte is present in sufficient quantity

and its performance as a fluoroplior when complexed with the target analyte is not adversely impacted to an unacceptable degree. Also in this regard, it should be understood that fluoroplior performance primarily means availability for binding reaction or the like with the target analyte to foπn a complex as discussed above, wavelength of the fluorescence peak, i.e., the wavelength (or more typically the wavelength range) of a fluorescence peak emitted by the complex upon exposure to excitation illumination, and other such properties or performance characteristics.

[78] The complexing reactions utilized in the systems disclosed here are not limited to immunological interactions, such as antigen-antibody or hapten-antibody interactions, but may instead or in addition be any other interactions of biological affinity, or other reactions, such as lectin-sugar or active ligand-receptor interactions, etc. Various other suitable interactions will be apparent to those skilled in the art given the benefit of this disclosure.

[79] As indicated above, conjugated nanocrystal fluorophores employed in the systems disclosed here preferably have a semiconductor nanocrystal core. Such a nanocrystal core or quantum dot exhibits characteristic fluorescence, absorption and time domain optical properties, where those properties are largely determined by, and so with adequate precision can be selected by, the size and composition of the nanocrystal core. Suitable conjugated nanocrystal fluorophores for use in the systems disclosed here can be prepared in accordance with known techniques. The fluorescence of the nanocrystal core is reversibly quenched by a coating, typically an organic coating. In certain exemplary embodiments the coated semiconductor nanocrystal is stable and suitable to be suspended in aqueous solution. A conjugated nanocrystal fluorophor of a device or method disclosed here will typically employ a nanocrystal core between 2

nm and 10 ran in diameter and composed, for example, of Group II- VI, III- V, or IV- VI semiconductors. Fluorescence typically will include a narrow, well defined peak within the wavelength range 300 nm to 2500 nm, although water stabilized nanocrystals preferred for use in conjugated nanocrystal fiuorophores for certain embodiments of such devices and methods may fall within a somewhat more limited wavelength range, e.g., within the wavelength range 300 nm to 2500 nm. Coated semiconductor nanocrystals suitable for use in preparing conjugated nanocrystal fiuorophores for devices, methods and systems disclosed here are commercially available, including, for example, EviTags® available from Evident Technologies. The coated semiconductor nanocrystals are terminated with a suitable functional group, such as, for example, the well-known functional groups or moieties of carboxylic acid, amine, etc. Once functionalized, the coated semiconductor nanocrystals can be easily conjugated to a bio-affinity molecule or moiety corresponding to the biomolecular analyte to be determined by the device or method. Such conjugated nanocrystal fiuorophores suitable for use in systems disclosed here can be prepared using known techniques from EviTags® or other such commercially available materials or can be obtained commercially, for example, from Evident Technologies, Troy, NY, and Antibodies, Inc., Davis, CA.

[80] In accordance with certain exemplary embodiments the coating on a conjugated nanocrystal fluorophor is wholly or partly removed, thereby unquenching the nanocrystal core, when the conjugated nanocrystal fluorophor is complexed with one or more of the corresponding biomolecular analytes. Exposing such a complex to excitation illumination will result in detectable fluorescence at the appropriate wavelength, meaning at the wavelength or range of wavelengths determined in

advance to be characteristic of the unquenched nanocrystals being employed for that particular target analyte. Detecting fluorescence at such pre-determined wavelengths, therefore, is a positive test result, indicating the presence of the biomolecular analyte of interest in the tested biological sample. In that regard, as used here and in the appended claims, a "biomolecular analyte" is any molecule or moiety (or derivative thereof as discussed above), found in a biological sample fluid, e.g., human or other mammal or other animal fluid, such as blood, plasma, serum, saliva, urine, semen, lymphatic fluid, cerebrospinal fluid, tears, milk, tissue homogenate, cell lysate, moisture in exhaled breath, etc. Exemplary analytes include proteins, peptides, polypeptides, lipids, polysaccharides, carbohydrates, nucleotides, nucleosides, nucleic acids, nucleic acid analogs, haptens, antigens, antibodies, DNA, RNA, and the like.

[81] In accordance with certain exemplary embodiments, determination of an analyte may be simply assaying whether a particular biomolecular analyte is present, i.e., whether a detectable quantity of such biomolecular analyte is present or a quantity above some threshold. The system may generate an electronic signal indicative of the result of the analysis, or a visual signal, etc. In accordance with certain exemplary embodiments the method or device is operative to determine an analyte quantitatively. Optionally systems that perform a quantitative assay of the analyte in the sample fluid provide a numerical output, e.g., a quantitative value corresponding to the absolute quantity of the analyte in the fluid sample tested or a quantitative value corresponding to the amount of analyte per unit volume, etc. hi certain embodiments of the systems disclosed here, the device or method is operative to assay a pre-determined quantity of biological or other sample fluid. For example, the biological fluid sample access to the reaction site may comprise a fixed volume chamber, e.g., a capillary chamber.

The reaction site may comprise a fixed volume chamber. A fluid delivery device, such as a wick, capillary or the like, may deliver the biological sample (with or without pre-treatment of the sample, e.g., by addition of buffer, other reactants, etc.) in limited or predetermined amount or at a fixed or pre-determined flow rate, whereby a pre-determined quantity of biological fluid is tested, optionally by time-limiting the assay. Other suitable means for assaying a pre-determined quantity of biological fluid will be apparent to those skilled in the art given the benefit of this disclosure.

[82] As used here and in the claims, the term "complexed" means that the conjugated nanocrystal fluorophor is reacted with or otherwise coupled or joined with the corresponding biomolecular analyte, optionally via an intermediary linking molecule or moiety, e.g., via a bi-specific antibody, so as to unquench the conjugated nanocrystal fluorophor. The term "unquench" means to render a conjugated nanocrystal fluorophores fluorescent under exposure to excitation light, i.e., laser or other light comprising one or more wavelengths effective to cause the nanocrystal to fluoresce. As described, the conjugated nanocrystal fluorophores preferred for use in the systems disclosed here comprise a normally fluorescent semiconductor nanocrystal core reversibly quenched with an organic coating. That is, the core is naturally fluorescent under excitation light, but the coating of the conjugated nanocrystal fluorophor is effective to quench the nanocrystal, i.e., to prevent such fluorescence in the use environment. The coating provides functional groups, e.g., up to around 100 functional groups or more per coated nanocrystal, for conjugation with a binding entity, e.g., a molecule or moiety having a suitable binding site (e.g., an aptamer, oligonucleotide, antibody, etc.). Upon one or more of the binding entities of a conjugated nanocrystal fluorophor being complexed with the target analyte (either

directly with the target analyte or with a product of the target analyte and an intermediary, e.g., a bi-specific antibody discussed elsewhere herein), fluorescence is unquenched at least in the area local to the participating binding entity. That is, at least the local area of the coating is removed from the nanocrystal core or disabled in its quenching function, thereby rendering the complex fluorescent. The fluorescence of an unquenched conjugated nanocrystal fluorophor can be detected, thereby determining the presence of the target analyte. In certain exemplary embodiments determining the analyte includes quantifying the analyte in the biological fluid sample.

[83] The biological fluid sample access for sample fluid to the reaction site may be any of numerous different forms, means, or the like, for example, a simple opening through a wall of the hand-held device containing the reaction site, or a gated or otherwise openable and closable opening, a funnel-shaped entryway, a capillary feed tube from a threshold sample application zone to the reaction site, etc. Especially in certain exemplary embodiments wherein the reaction site contains the conjugated nanocrystal fluorophores in a dry state immobilized to a substrate, e.g., a fibrous or plate-like substrate etc., the reaction site may simply be exposed for contact with the biological fluid sample. That is, in such embodiments the biological fluid sample access to the reaction site may be provided by direct exposure of the reaction site. Alternatively, the reaction site may have indirect exposure, e.g., via a fluid communication pathway such as a wicking member, e.g., a bibulous member, capillary or other feed line from an exposed sample application zone to the reaction site. As used here and in the claims, the term "operator-selectable condition" refers to the position, setting or other condition of the biological fluid sample access to the reaction site in certain

exemplary embodiments wherein the access has more than one condition. More specifically, the operator-selectable condition is a condition of the port which is selectable by the operator of the device or system or by the person or equipment performing the method. For example, in certain exemplary embodiments the biological fluid sample access to the reaction site is not in fluid communication with the reaction site when the device is in storage before use. In certain exemplary embodiments it is not in fluid communication when the biological sample is introduced via the port. In certain exemplary embodiments the biological sample contacts a pre-conditioning reagent, e.g., a bi-specific reactant as discussed further elsewhere in this disclosure, optionally in a pre-reaction chamber that is segregated from the main reaction chamber containing the conjugated nanocrystal fluorophores. In using certain such embodiments, the operator, typically a physician, nurse, lab technician or the like, removes a barrier or opens a fluid flow channel or otherwise establishes fluid communication to the main reaction chamber during running of the assay.

OTHER EXEMPLARY EMBODIMENTS

1. Embodiments Using Diabody Conjugates

[84] In accordance with certain exemplary embodiments of the fluorescence-based systems disclosed here for determining a biomolecular analyte in a biological fluid sample, the presence or absence of specific analyte(s) in the sample is assessed using a noncompetitive, homogeneous fluorescence-based diagnostic system involving

interactions between the analyte of interest, a bispecific antibody, and EviTag® semiconductor quantum dots. The bispecific antibody preferably is generated to

include an affinity domain binding site (e.g., at one end of the molecule) as well as an

anti-EviTag® conditional-affinity domain. In its unbound state, the EviTag® remains

quenched due to the presence of conducting polymer on the surface of the quantum dot. Upon addition of sample the target molecule will bind to the analyte-specific

affinity domain of the diabody. In this state the anti-EviTag® affinity domain is

enabled to bind the quenched EviTag®, resulting in the displacement of conducting

polymer from the quantum dot core, and consequent unquenching. Light emission can then be read at the appropriate wavelength via the observation access, e.g., a suitable hole or window in the housing of a hand-held device containing the test site. Additional embodiments employing diabody techniques will be apparent to those skilled in the art given the benefit of this disclosure.

2. FRET (Fluorescence Resonance Energy Transfer)

[85] In accordance with certain exemplary embodiments of the fluorescence-based systems disclosed here for determining a biomolecular analyte in a biological fluid sample, the presence or absence of specific analyte(s) in the sample is assessed using the combined technologies of fluorescence resonance energy transfer (FRET) and conjugated semiconductor quantum dots, such as, e.g., EviTag® quantum dots available from Evident Technology (Troy, New York), as the conjugated nanocrystal fluorophores. In such embodiments a competitive homogeneous assay is performed

using an analyte-specific antibody or aptamer or the like conjugated to an EviTag® as

the FRET donor, the target molecule labeled with an organic fluorophor as acceptor. In the absence of the specific target molecule in the sample, the antibody/aptamer-

labeled EviTag® and the organic fluorophor-labeled target specifically combine.

Their close proximity allows for efficient energy transfer to occur between the

quantum dot (EviTag®) donor and the organic dye acceptor. Competitive binding of

the analyte of interest will result in the displacement of the fluorophor-labeled target and a subsequent reduction FRET (due to decreased proximity). This is reflected by a decrease in light emission from the acceptor fluorophor molecule and a simultaneous increase in light emission from the donor quantum dot when the signal is measured at the appropriate wavelengths. Further in this regard, FRET techniques suitable for application to the assay systems disclosed here are taught, for example, in the following cited material: Qin Q. et al., "Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin," Clin. Chem. 49(7): 1105-1113, (2003); Analysis of STAT3 (signal transducer and activator of transcriptions) dimerization; Rapid detection and quantitation of hepatitis B virus DNA; Hepatitis C genotype determination; Rapid detection of factor XIII Val34Leu polymorphism; detection of epitope-specific characterization of anti-platelet antibodies; Youn HJ, Terpetschnig E, Szmacinski H, Lakowicz JR, "Fluorescence energy transfer immunoassay based on a long-lifetime luminescent metal-ligand complex," Anal. Biochem. 232:24-30 (1995); Blomberg K, Hurskainen P, Hemmila I, "Terbium and rhodamine as labels in a homogeneous time-resolved fluorometric energy transfer assay of the beta subunit of human chorionic gonadotropin in serum," Clin. Chem. 45:855-864 (1999); Ueda H, Kubota K, Wang Y, Tsumoto K, Mahoney W ₅ Kumagai I, Nagamune T, "Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay)," Biotechniques 27:738-742 (1999); Selvin PR, "The renaissance of fluorescence resonance energy transfer," Nat. Struct. Biol. 7:730-734 (2000).

[86] The presence or absence of specific analyte in the sample is assessed in certain exemplary embodiments using an antibody/aptamer-based fluorometric probe

comprising primary antibody/aptamer conjugated to EviTag® quantum dots as the

conjugated nanocrystal fluorophor for the target analyte. In its unbound state, the

EviTag® remains quenched due to the presence of conducting polymer on the surface

of the quantum dot. Upon selective binding of the analyte of interest to the

antibody/aptamer conjugated to the EviTag® the conducting polymer is, at least in part, released or removed from the nanocrystal core, resulting in the

unquenching/activation of the EviTag®. Upon exposure to excitation light,

fluorescent emission can then be read at the appropriate wavelength.

[87] In accordance with another aspect, a fluorescence-based assay system comprises at least one hand-held device or method as disclosed above for determining at least one biomolecule analyte in a biological fluid sample and a data collection and storage subsystem operative to capture test results of such hand-held device(s). In certain exemplary embodiments such data collection and storage sub-system comprises a general purpose data processor, e.g., a general purpose microprocessor, laptop or desktop computer, or the like, and software loaded on such hardware for controlling the receipt and storage of test data either directly from the hand-held device, e.g., wirelessly or via the internet or other wired connection, or indirectly, e.g., with intermediate collection and storage of the date by other suitable hardware. In accordance with certain exemplary embodiments auxiliary data relating to the test results is also captured, i.e., collected and stored, e.g., patient information such as patient identification information, patient history or condition information and the like, data identifying the serial number or other identifying infoπnation for the

specific hand-held unit used in the test, environmental data or the like, etc. Certain exemplary embodiments further comprise data distribution capabilities, for example communication means and associated control software for distribution of test results back to the point-of-care provided whence the data originated and/or to other recipients, such as medical insurance providers, consulting physicians, survey or group study organizations, etc. In accordance with certain exemplary embodiments, systemic product and usage monitoring capability is provided. For example, capability optionally is provided, such as communication and system control software, to capture test results from each hand-held device and to compare such results to some or all like units deployed in the past. For example, capability optionally is provided, such as communication and system control software, to track supply volumes by user facility and to generate and deliver messages relating to inventory levels, for example, to recommend re-stocking requirements to a user facility such as a medical office, community clinic, hospital or the like. For example, capability optionally is provided, such as communication and system control software, to determine whether a handheld unit or other component of the system is used properly and in a suitable use environment (e.g., temperature, etc.) In accordance with certain exemplary embodiments, system update capability is provided. For example, capability is provided, such as communication and field programmable system control devices and software, to automatically or on demand deliver new software versions to all or certain computer units of the data collection and storage sub-system. For example, capability optionally is provided, such as communication and system control software, to deliver new or different operating parameters or control software to hand-held units equipped with a microprocessor or other controller and/or memory.

[88] While certain particular embodiments of the invention have been illustrated and described, it will be apparent to those skilled in the art that numerous modifications and additions can be made without departing from the true spirit and scope of the invention. Accordingly, it is not intended that the invention be limited except in accordance with the terms of the following claims. In the following claims, definite and indefinite articles such as "the," "a," "and," and the like, in accordance with traditional patent law and practice, mean "at least one." Thus, for example, reference above or in the claims to "a test site containing anti-conjugated nanocrystal fluorophores for a biomolecular analyte" means at least one test site each containing anti-conjugated nanocrystal fluorophores for at least one biomolecular analyte. Optionally, any or all of such one or more test sites may contain anti-conjugated nanocrystal fluorophores for two or more different biomolecular analytes of interest.

[89] In general, unless expressly stated otherwise, all words and phrases are used above and in the following claims have all of their various different meanings, including, without limitation, any and all meaning(s) given in general purpose dictionaries, and also any and all meanings given in science, technology, medical or engineering dictionaries, and also any and all meanings known in the relevant industry, technological art or the like. Thus, where a term has more than one possible meaning, all such meanings are intended to be included for that term as used here. In that regard, it should be understood that if a device, system or method has the item as called for in a claim below (i.e., it has the particular feature or element called for, e.g., a test site containing anti-conjugated nanocrystal fluorophores for a biomolecular analyte), and also has one or more of that general type of item but not as called for (e.g., it has one or more other test sites that do not contain anti-conjugated nanocrystal

fluorophores for a bioniolecular analyte), then the device, system or method in question satisfies the claim requirement. Those one or more extra items are simply ignored in determining whether the device, system or method in question satisfies the

claim requirement.