| US4968601A | 1990-11-06 | |||
| US5482830A | 1996-01-09 | |||
| US5541057A | 1996-07-30 |
The ability to identify a patient's infection is generally limited to the century-old technique of"swab and culture."Samples of potentially infectious agents taken from a patient are placed in an enrichment medium, grown, and then identified using biological assays that measure specific metabolic profiles. Viruses and rickettsia are detected through techniques such as immunoassays that detect viral antigen or Polymeruse Chain Reaction ("PCR") that detects production of virus genes. These methods require laboratory facilities, expertise in culturing methods, expertise in interpretation of results, and long incubation periods.
SUMMARY An abbreviated description of the preferred embodiment (s) of the invention is summarized here to highlight and introduce novel aspects of the present invention.
Simplifications and omissions may be made in this summary. Such simplifications and omissions are not intended to limit the scope of the invention.
The present invention relates to a method and apparatus for early detection of infectious agents. The preferred embodiment senses changes in the coupling ratio between two coupled optical fibers caused by changes in the environment within the evanescent fields of the fibers. Changes in the evanescent fields are caused by a binding interaction between receptor compound coupled to the optical fibers and an infectious agent of interest.
Another aspect of the present invention is the use of a receptor compound coated on the surface of an optical fiber coupler wherein the receptor compound selectively binds with influenza virus.
Yet another aspect of the present invention is the coupling of a small chemical molecule to an optical fiber for use in an infectious agent detection apparatus.
An additional aspect of the present invention is the use a modular detector apparatus comprising a removable probe cartridge and a base unit that includes a display.
Another aspect of the present invention is the use of a reference coupler to eliminate noise and interference.
BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiment (s) of the invention will be discussed below with reference to attached drawings.
FIG. 1 illustrates a packaging arrangement for an apparatus consisting of a removable probe cartridge and a base unit.
FIG. 2 schematically illustrates components of a probe cartridge consisting of seven components: (1) an optical light source; (2) a signal splitter; (3) a sample probe and an optical reference coupler; (4) photodiodes; (5) transimpedance amplifiers; (6) analog to digital converter; and (7) an electronic circuit to capture, analyze and display data.
FIG. 3 illustrates the two halves of a single probe.
FIG. 4 illustrates a preferred receptor compound for influenza virus.
FIG. 5 illustrates a preferred receptor compound as prepared for coupling to an optical fiber.
FIG. 6 illustrates a preferred crosslinking compound.
FIG. 7 illustrates an embodiment of a process for binding a receptor compound to an optical fiber.
FIG. 8 illustrates a post-binding chemical process for removal of protecting groups.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT (S) Fig. 1 illustrates one suitable packaging arrangement for an infectious agent detection apparatus. The apparatus includes a base unit 101 and a removable probe cartridge 103. The base unit 101 has an electronic touch screen display 105 and a receptacle 107 for receiving the probe cartridge 103. The probe cartridge 103 has an injection port 111 suitable for receiving a fluid sample to be tested, e. g., by injection with a syringe. The probe cartridge 103 has at least one electrical connector 109 which mates with a counterpart connector (not show) inside the receptacle 107 of the base unit 101. The depth of the probe cartridge 103 is greater than the depth of the receptacle so that the injection port remains accessible when the probe cartridge electrical connector 109 is seated within the base unit 101.
In one version, the probe cartridge 103 receives electrical power for internal electronic circuitry through the electrical connector 109. Alternately, the probe cartridge 103 may have an additional receptacle (not shown) for a battery (not shown). Furthermore, the probe cartridge 103 and base unit 101 may have additional connectors (not shown) through which the base unit 101 may supply fluids to, and potentially receive fluids from, a probe cartridge 103.
Fig. 2 schematically illustrates functional components of a probe cartridge. An optical light source 121 launches light into an optical fiber 123. A one-by-n ("1 x n") splitter 125 divides the light into multiple, preferably two, light signals of equal amplitude, each in separate optical fibers 127,129. A sample probe 131 and a reference coupler 133 each receives a light signal from one of the optical fibers 127,129. Each of the sample probe 131, and reference coupler 133 has a pair of output fibers 135,137,139,141. Each of four photodetectors 143,145,147,149 receives light from one output fiber 135,137,139,141.
Each of four transimpedance amplifiers 151,153,155,157 amplifies the outputs of the photodetectors 143,145,147,149. A multi-input A/D converter 159 samples the output signals from the transimpedance amplifiers 151,153,155,157 and provides digital measures of signal amplitude to a control and communication circuit 161. The control and communication circuit regulates sampling and provides a digital interface, such as an IEEE 488 interface through the connector 109 (Fig. 1) of the probe cartridge.
The sample probe 131 and reference probe 133 each receives a fluid sample to be tested. The fluid sample may be injected through an injection port 163 into a storage bladder
165. The control and communications circuit 161 operates a valve 167 to allow a controlled volume of sample fluid to flow into the sample probe 131 and reference coupler 133.
The optical light source 121 may be a laser or light emitting diode (LED) driven by a controller and a thermoelectric (Peltier) cooler. The optical light source preferably generates light at a wavelength of between 780 and 1300 nm. Laser sources such as Seastar PT-50-780, Seastar PT-450-840, Corning SMF-28, Mitsubishi FU-43SLD-IAT, or Laser Diode, Inc., LAT E-300 may be used for bench testing, however, production probe cartridges would preferably be low cost LED's similar to ones used in the communication industry. Optical fibers may be Corning Flexcore 850 single mode waveguide fiber, or Corning SMF-28.
Photodiodes may be Melles-Griot 13DSI011 (for light wavelengths below 900nm) or Germanium Power Devices Corp. GE-7 (e. g. for 1300 nm wavelength light.) Transimpedance amplifiers may be Melles-Groit 13 AMP003. The light source 121, A/D converter 159, and control and communication circuits may draw power from a battery (not shown) in the probe cartridge or through the probe cartridge connector 109 (Fig. 1).
Fig. 3 illustrates two halves 203,205 of a single probe 201. A first clamshell housing 211 is made from a straight piece of glass tubing from which half has been ground away to leave a"C"-shaped cross section. Epoxy plugs 217,219 at opposite longitudinal ends of the first clamshell housing 211 hold short lengths of hollow glass access tubes 213,215. The epoxy plugs 217,219 extend only partly along the axis of the first clamshell housing 211 so as to leave a central cavity 221. The access tubes 213,215 lie parallel to the axis of the first clamshell housing 211 and extend completely through the epoxy plugs 217,219 so that fluid can pass through the access tubes 213,215 to the central cavity 221.
A second clamshell housing 231 is made from a straight piece of glass tubing from which half has been ground away to leave a"C"-shaped cross section. The first and second clamshell housings 211,231 should be formed so that they may be joined to form a single, substantially cylindrical unit. Epoxy plugs 241,243 at opposite longitudinal ends of the second clamshell housing 231 hold a pair of coupled optical fibers 233,235. The epoxy plugs 241,243 extend only partly along the axis of the second clamshell housing 231 so as to leave a central cavity 239 that is substantially complementary to the central cavity 221 of the first clamshell housing 211. The optical fibers 233,235 have been drawn and twisted in a waist region 237 which is position in the central cavity 239 of the second clamshell housing 231.
The clamshell tubing may be three inches long with one-quarter inch diameter, however, longer or shorter lengths, and longer or shorter diameter tubing may be used. The central cavities 221,237 may be one-and-a-half to two inches long (for a three-inch clamshell housing), or longer or shorter. Preferably, the central cavities will have a volume on the order of ten milliliters (for laboratory use) or one milliliter (for clinical use). The glass tubing of the clamshell housings 211,231 and of the access tubes 213,215 should be silated prior to assembly. Silation will protect these glass portions from other chemical reactions that will be performed on the glass of the optical fibers as discussed more fully below.
The optical fibers 233,235 should be drawn and twisted to form a fusion joint as generally described in U. S. Patent No. 5,494,798 (the'798 patent) which is incorporated herein by reference in its entirety. Such a fusion joint couples the fibers so that the evanescent field of each fiber permeates at least a portion of the other fiber. In such a configuration, a portion of the light energy injected into one fiber will propagate to the other fiber.
The first and second halves 203,205 are joined to form a single coupler which may be used as a splitter (item 125, Fig. 2), or further processed to form a reference coupler (item 133, Fig. 2) or a probe (item 131, Fig. 2). Couplers may be acquired from Empirical Technologies Corporation, of Charlottesville, Virginia or from the inventors named in the '798 patent.
A coupler may be used as a probe after a receptor compound is applied to the fusion joint 237 of the twisted and drawn optical fibers 233,235. Receptor compounds for detection of infectious agents may be chosen from existing compounds known to specifically interact with an infectious agent to be detected or may be independently designed to exhibit specific binding. Reference couplers are prepared in a manner similar to probes, except that the compound coupled to the fusion joint does not exhibit specific binding to the infectious agent to be detected.
When assembled into a probe cartridge, one access tube 211 of a probe connects through the bladder (item 167, Fig. 2) to the injection port (item 163, Fig. 2), so that a fluid sample injected into the injection port may flow into the central cavity 239 and be exposed to the waist region 237 of the coupled fibers 233,235. The other access tube 215 may be connected to a drain reservoir (not shown) which accumulates excess fluid. Alternatively, fluid paths may be provided within a probe cartridge to both the injection port and to
connectors on the housing of the probe cartridge, which would in turn connect to the base unit (item 101, Fig. 1). In this configuration, the base unit 101 regulates the flow of fluid to carry a sample from the injection port to the probes. The base unit 101 could later regulate the flow of a purging fluid to remove sample from a probe cartridge for disposal. Such purging could also be used to clean the probes in preparation for receiving a new sample.
Fig. 4 illustrates a structure for a preferred receptor compound for detecting human influenza virus. The receptor compound has a structure which includes an aromatic ring with three functional attachments. A first functional attachment (A) is a carboxylic acid group. A second attachment (B) is a guanidine group. The third attachment (C) is an amide. The first and second functional attachments (A) and (B) present a configuration that exhibits very strong, specific binding to the hemagglutinin (HG) glycoproteins that comprise up to 90% of the surface proteins of the influenza virus capsule. The third functional attachment (C) may be modified for attaching receptor compound to an optical fiber.
A sample probe (item 131, Fig. 2) would use the compound of Fig. 4, while a reference coupler 133 would use no compound or a compound exhibiting no, or relatively weak binding. Detection is based on the differentiation between strong, specific binding (in the probe) and weaker, non-specific binding (in the reference coupler). For example, sialic acid is known to bind to the HG glycoproteins of the influenza virus. Sialic acid exhibits non-specific binding as well as specific binding. However, the receptor compound in Fig. 4 binds about 10,000 times more strongly than sialic acid. The reference coupler would thus have similar activity as the probe with respect to light and fluid, and exhibit a weaker, non- specific binding interaction with the virus.
The process of coupling the receptor compound or the compound used on a reference coupler to the optical fibers proceeds through four general steps. These steps will be described with respect to coupling the receptor compound. Fewer or different steps may be required for coupling the compound used on the reference coupler to the optical fibers.
In a first step, a receptor compound preparation process prepares the receptor compound for a binding reaction with the optical fiber and protects certain functional attachments from the coupling reaction. Second, a fiber preparation process prepares the optical fibers for a binding reaction with the receptor compound by attachment of a crosslinking compound. Third, a binding reaction couples the receptor compound to the crosslinking compound on the optical fibers. Fourth, a post-binding process removes the
protecting groups from the functional attachments of the receptor compound. The second, third, and fourth steps may be performed within a previously-fabricated probe 201. Each step is discussed in more detail below.
The receptor compound preparation process synthesizes and prepares the receptor compound for binding. Fig. 5 illustrates the receptor compound at the conclusion of the preparation process. The receptor compound preparation process attaches a methyl ester protective group to the carboxylic acid functional attachment (A). The preparation process also attaches tert-butoxycarbonyl protective groups (Boc) to the guanidine functional attachment (B). The receptor compound preparation process also attaches a carboxylic acid group through an alkyl chain to the third attachment (C). The alkyl chain may vary in length, but would preferably have a length of between 1 and 20 carbon atoms, more preferably between 1 and 10 carbon atoms, and most preferably would have about three carbon atoms.
In the form shown in Fig. 5, the exposed carboxylic acid group on the third attachment (C) is left unprotected in order to participate in the binding reaction to the optical fiber, while the first and second functional attachments (A) (B) remain protected. If the compound to be coupled to the reference coupler does not contain the guanidine and the carboxylic acid functional groups, fewer or different protection steps may be used. For example, if sialic acid is used as the compound on the reference coupler, the carboxylic acid may be protected as the methylester similar to the receptor compound. Standard protective methodologies are available to protect the hydroxyl groups on the sialic acid.
The fiber preparation process attaches a crosslinking compound to the matrix of the optical fiber. Fig. 6 illustrates a preferred crosslinking compound, which includes a siloxane functional group (D) and an amine functional group (E) joined through an alkyl chain. The alkyl chain length may vary, but would preferably have a length of between 1-10 carbon atoms, more preferable between 1 and 5 carbon atoms, and most preferably would have three carbon atoms. The siloxane functional group (D) attaches to the optical fiber through standard hydrolysis and condensation reactions, which leaves the amine group (E) available for coupling with the carboxylic acid group on the third attachment (C) of the receptor compound and similar functional group on the compound to be attached to the reference coupler.
Fig. 7 illustrates the binding reaction. The binding reaction exposes the prepared optical fibers to prepared receptor compound in the presence of dicyclohexylcarbodiimide
(DCC). The amine-terminated optical fiber then undergoes nucleophilic substitution with the carboxylic acid group on the receptor compound in the presence of DCC and dimethylaminopyridine (DMAP). For example, the protected receptor compound (Fig. 5) in anhydrous dichloromethane may be added to the coupler within the probe of Fig. 3 at 0° C.
Alternatively, the probe could be placed in a reaction vessel to which the solution of amine is added. Ideally, a stoichiometric amount of protected receptor compound relative to the amine group on the optical fiber should be used. To this is added a small amount (approximately 0.1 equivalents) of DMAP and one equivalent of DCC. After addition is complete, the temperature may be allowed to rise to room temperature. The probe, including the fiber optics, may be removed from the reaction vessel (or emptied) and rinsed with water followed by rinsing with dichloromethane. The compound to be attached to the reference coupler may be attached using an identical or modified set of reactions.
The post binding process deprotects the first and second functional attachments of the receptor compound. Fig. 8 illustrates the post-binding chemical processes. The protecting groups are removed using standard procedures well known in the chemical arts. For example, to remove the BOC group, a 1: 1 (v/v) solution of trifluoroacetic acid (TFA) in dichloromethane may be added to the probe. Alternatively, the probe may be placed in a vessel containing the TFA/dichloromethane solution. Reaction should be continued for 16-24 hours. The probe may be emptied (or removed from the reaction vessel) and dried.
The methyl ester may be removed by adding a solution of 1 N NaOH to the probe (or adding the probe to a vessel containing the NaOH solution). After agitating for 2-3 hours at room temperature, dilute HC 1 is added until the solution is slightly acidic (pH= 3-5). The probe is then emptied (or removed from the reaction vessel), rinsed with water and then rinsed with ethanol. The probe is now prepared for use. The compound attached to a reference coupler may require different deprotection steps.
Basic operation of the detection apparatus will be described with reference to Figs. 1, 2, and 3. An operator would turn on the base unit 101 (Fig. 1) and enter patient information and commands through the touch screen 105 to initialize an analysis sequence. The operator would seat a probe cartridge into the base receptacle 107 and enter a command to initiate a calibration sequence. The programmed computer in the base 101 communicates with the probe cartridge 103 through the electronic port 109. About ten times each second, the programmed computer acquires digital measurements of the light amplitude from outputs of
all four transimpedance amplifiers 151,153,155,157. Two of the transimpedance amplifiers 151,153 monitor the light amplitudes from the fibers of the sample probe 131, which has been treated with receptor compound. For the purpose of the discussion below, transimpedance amplifier signals from the two outputs of the sample probe 131 will be designated"A"and"B."Transimpedance amplifier signals for the two outputs of the reference coupler will be designated"C"and"D." During a programmable baseline measurement period that may range from several seconds to several minutes, the programmed computer acquires data from the amplifiers 151, 153,155,157 and establishes several baseline measurements. First, the programmed computer computes a baseline normalized sample probe coupling ratio (BNPR) as: BNPR (0) = (A/ (A+B))/C 1, where Cl is a normalizing constant derived from samples of many probes. For example, Cl may be the average or median coupling ratio taken from a large sample of probes taken from a single manufacturing lot.
Second, the programmed computer computes baseline normalized reference coupling ratio (BNRR) as: BNRR (0) = (C/ (C + D))/C2, Where C2 is a normalizing constant derived from samples of many couplers. For example, C2 may be the average or median coupling ratio taken from a large number of reference couplers from a single manufacturing lot. C 1 and C2 may be different, because probes and reference couplers will have been treated with different compounds and thus may have been subject to different processing conditions.
Third, the programmed computer 101 will compute a baseline detection value BDV (0) as: BDV = BNPR-C3 * BNRR. where C3 is a normalization constant used to adjust BDV to zero in the absence of sample.
Fourth, the base programmed computer will compute a baseline noise level VAR (0) as the variance of BDV over a programmable time period, e. g., one to three minutes.
After acquiring a minimum number of samples for computing VAR (0), the programmed computer will signal the operator that it is ready to receive a sample to be analyzed. The operator will acquire a sample to be tested, such as by swabbing a patient's nasal membranes or throat tissue with a cotton swab. The operator would wash the swab in a small quantity of transfer fluid, such as a PBS or other buffer solution and then inject the transfer fluid into the injection port 111 of a probe cartridge 103. The operator could obtain the sample before or while the base computer was computing the baseline values. The injection operation transports the transfer fluid into the bladder 165 (Fig. 2). The operator would then enter a command into the base unit 101 indicating that a sample has been injected.
The base computer operates the valve 167 to allow fluid sample to pass to both the sample probe 131 and to the reference coupler 133. The base computer continues to obtain measurements from the probe cartridge and continues to compute BNSR, BNPR and BDV at regular intervals during a programmable detection period which could last from about one to ten minutes. The base unit will signal to the operator that influenza virus has been detected if BDV changes by more than a multiple of the noise (VAR (0)) during the detection period, i. e., if : BDV (t)-BDV (0) > 2 * VAR (0).
In addition, the base computer may display a graph of BDV (t) with a grid overlay that would permit an operator to view data as a function of time.
The detection process relies on the coupling sensitivity of the fusion joint of the probe. The fusion joint is drawn to such a thin dimension that the evanescent field of one fiber permeates the other fiber. In such a configuration, some of the light energy carried in one fiber transfers to the other fiber.
The coupling ratio is affected relatively strongly by the wavelength of light and by the index of refraction of the optical fibers. The calibration method described herein eliminates the variability caused by differences in the refractive index of the optical fibers by measuring relative changes in coupling ratios between fibers treated with receptor compound and
reference fibers. Utilizing the same transfer fluid in both the probe and coupler eliminates variations caused by the liquid environment surrounding the couplers. Furthermore, by coating the reference coupler with a compound that exhibits weaker binding and non-specific binding to the analyte of interest, false positive results caused by non-specific binding and weak interactions may be reduced, thus increasing the sensitivity and the specificity of the detector. Variability in coupling ratios caused by the wavelength of light, as well as variability caused by other factors such as temperature, are also eliminated by measuring relative coupling ratios.
The apparatus described above can be adapted to automated sampling and testing.
For example, an operator could fill a multiplicity of sample vials with transfer fluid from several patients. An autoinjector, such as is commonly used in HPLC applications, could remove a predetermined amount of transfer fluid from a first sample vial. After the computer control signals that the calibration process is complete, the autoinjector would inject the sample through the injection port (item 163, Fig. 1), filling the bladder (item 165, Fig. 1).
The computer than monitors BDV (t) for a predetermined time period. At the end of the designated time period, a flushing solution from a reservoir would flush the probe and standard coupler with a predetermined volume of flushing solution sufficient to remove the first sample from the probe and standard coupler. The computer could then recalculate BNPR (0) and BNRR (0). If these are not within a predetermined range, for example 5% of the initial values before injection of the first sample, the flushing process is repeated until both BNPR (0) and BNRR (0) are within an acceptable range of the initial values. Once such a condition is met, the autoinjector removes transfer fluid from a second sample vial and the process repeats.
Data from each of the multiple samples could be stored separately within computer RAM or recorded as individual files on computer disc for later access. Upon retrieval of data for each sample stored in RAM or on disc, the computer displays data as for individual samples. Alternatively, the apparatus could print data for each sample as the detection process is completed.
Additional information may be found in U. S. Provisional Patent Application entitled, "Influenza Virus Hemagglutinin Ligand and Methods of Using the Same,"filed on or about June 28,1999 in the names of Ashraf Saeed, Shijia Yan, and Stephen Jonson, which is incorporated herein by reference in its entirety.
One or more preferred embodiments have been described to illustrate the invention (s).
Additions, substitutions, modifications, and/or omissions may be made to the preferred embodiment (s) without departing from the scope or spirit of the invention (s). It is the intent of the following claims to encompass all such additions, modifications, and/or variations to the fullest extent permitted by law.