OPTICAL RESONANCES IN DROPLETS IN A MICROCHANNEL

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001J This application claims priority from U.S. Provisional Patent Application Serial No. 60/808,768, which is hereby incorporated by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The U.S. Government has certain rights in this invention pursuant to Grant No. 5P42ES04699 awarded by the NlEHS Superfund Program. BACKGROUND

Field of the Invention (0003] This invention relates to the fields of chemistry and biology.

Description of the Related Art [0004] During the last decade, optical biosensors capable of detecting unlabeled macromolecules have become valuable tools in life sciences. Optimal biosensor design should be simple and sensitive. High sensitivity is of critical importance, since one must often detect only a few molecules. [0005] Recent studies have shown that optical resonances in microcavities, e.g., morphology dependent resonances (MDRs) or whispering galley modes (WGMs), can be used for biomolecule detection. It has been shown via protein detection by the shift of the resonant spectrum in a silica microsphere through the absorption of proteins on to the microsphere, occasioning a change in its optical properties and thereby offering a sensitive detection scheme. [0006] The resonances are generated when light, confined by internal reflection within the droplet, orbits near the droplet/oil interface and returns in phase after each revolution. The frequencies of these MDRs or WGMs are characterized by the number of wavelengths L within an orbit, are extremely sensitive to added dielectric material near the microparticles surface. It is estimated that an atomic thickness can lead to a detectable shift of a given resonance frequency.

[0007] Inclusions inside microcavities have a direct effect on the MDRs that are supported by fluorescence; modification of the resonances arises from changes in the scattering and local refractive index. Protein detection by the shift of the resonant spectrum in a silica

microsphere through the adsorption of proteins on to the microsphere has been demonstrated, occasioning a change in its optical properties and thereby offering a sensitive detection scheme. The effect of inclusions in a microcavity is of great interest from both the theoretical and experimental perspective. The size and number of inclusions will have a direct impact on the resonances inside the cavity through scattering and local refractive index changes. The radial position of the inclusion and its orientation with respect to the illumination source is also important. Experimental work has shown a variety of effects of inclusions in liquid microcavities. The change in the optical resonance spectrum can be observed at low concentrations where each microdroplet contains as little as one cell. [0008] One of the most common optical detection techniques used for bioassays is fluorescence detection. Some of these techniques use an affinity based energy transfer phenomenon called Fluorescence Resonance Energy Transfer (FRET). A non-radiative energy transfer occurs between a donor and an acceptor depending on the distance between them and the overlap of the emission spectrum of the donor and the excitation spectrum of the acceptor. The transfer efficiency depends inversely on the sixth power of the distance between the donor and the acceptor molecule leading to a powerful nanoscale (2-10 nm) measurement technique. The transfer efficiencies for a wide variety of fluorescent dyes have been studied. Non-fluorescent quenchers such as QSY can also be used as efficient acceptors (donors) because of their availability in a wide spectrum range and the absence of any intrinsic fluorescence emission. FRET can be used as an efficient detection tool for fluoroimmunoassay applications. Antibody and antigen pairs can be tagged with FRET pairs, e.g., antibody with a fluorescent dye and antigen with its FRET pair; and used in different immunoassay formats including competitive assays. The binding reaction can be observed as a simultaneous decrease in the donor signal and an increase in the acceptor signal due to the energy transfer between the fluorescent dyes when they are in close proximity. If a non- fluorescent dye (quencher) is used as an acceptor, then only the decrease in the donor signal is observed relative to the reaction between the antibody-antigen.

(0009] Microdroplets have several advantages for performing FRET based immunoassays. The first advantage is the small sample volume that is presented in the micron-sized droplets used in the experiments. The second advantage is the elimination of non-specific binding to the sample container walls. In known inkjet applications, the droplets are airborne and thus only interact with laser light. Another advantage arises from the optical cavity formed by the microdroplets. The energy transfer can be enhanced due to the morphology dependent

resonances captured inside the microdroplet. The emission from donor molecules can be efficiently coupled to these resonant modes, enhancing the field strength at those wavelengths. The acceptor molecules can be excited through these resonances without being in the close proximity of the donor molecule, which typically is a requirement for FRET. Thus a more efficient energy transfer mechanism can be established and used to detect a wide variety of binding reactions.

[0010] Known methods of establishing and using the microdroplet resonances include droplets falling in air that were created by an inkjet printer and solid silica spheres that are impregnated with a fluorescent material. However, the free falling droplets are subject to evaporation and movement by air currents. They also move very quickly. An analyte of interest cannot be introduced into the solid microspheres.

[0011] The present invention addresses these and other limitations of the prior art by providing microdroplets formed in a microchannel for establishing and using microdroplet resonances, which can be used for detecting an analyte of interest.

SUMMARY OF THE INVENTION

[0012] The present invention is defined by the following claims, and nothing in this section should be taken as a limitation on those claims.

[0013] In one aspect, the present invention comprises a novel method using a microchannel for forming aqueous droplets that enable internal reflection within the droplets sufficient to support morphology dependent resonances. The morphology dependent resonances can be used for detecting an analyte of interest within the droplets according to various known methods.

[0014] Aqueous microdroplets comprising glycerol and a fluorescent dye have been formed in a microchannel that contains a low refractive index oil according to one embodiment.

When the droplet is excited by a pulsed laser, strong resonances and lasing are established inside the droplet.

[0015] The microdroplet acts as a high quality factor optical cavity that supports morphology dependent resonances (MDRs), also known as Whispering Gallery Modes (WGMs). Enhanced radiative energy transfer, for example, through these optical resonances can be utilized as a transduction mechanism for chemical and biological sensing. In one application of the invention, enhancement in radiative energy transfer is observed when a donor/acceptor pair is present in the resonant medium of a microcavity. Strong coupling of acceptor emission

into optical resonances shows that the energy transfer is efficiently mediated through these resonances.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0016] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0017] Figure 1 illustrates an experimental setup for optical resonance experiments in microdroplets according to one embodiment. [0018] Figures 2A and 2B illustrate a microchannel without and with droplets, respectively.

[0019] Figure 3 illustrates a laser causing optical resonances within a microdroplet according to one embodiment.

[0020] Figure 4 shows the suppression of resonance peaks when the E-CoIi cells are added to a fluorescent solution according to one embodiment. [0021] Figure 5 shows ten consectutive spectra from cell detection experiments in microdrolets.

[0022] Figure 6 shows Biotin-4-fluorescein (Absorption/Emission maxima 495/519 nm) and streptavidin-Alexa Fluor 555 (Absorption/Emission maxima 553/568 nm) interaction

(Emission spectra at 485 nm excitation). [0023] Figure 7 shows 40 nm biotinylated fluorescent polystyrene beads

(Absorption/Emission maxima 505/515 nm) and streptavidin-Alexa Fluor 555 interaction

(Emission spectra at 485 nm excitation).

[0024] Figure 8 shows biotinylated fluorescent bead and streptavidin-Alexa Fluor 555 binding reaction in microdroplets. [0025] Figure 9 shows acceptor (Streptavidin-Alexa Fluor 555) emission due to donor

(biotinylated fluorescent bead) excitation through FRET as a result of the specific binding reaction in microdroplets. This figure shows the acceptor emission spectra in the 550-600 nm range and is enlarged from Figure 8.

[0026] The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Advantages and Utility

[0027] Briefly, and as described in more detail below, described herein are methods, and apparatus for forming aqueous droplets that enable internal reflection within the droplets sufficient to support morphology dependent resonances. The morphology dependent resonances can be used for detecting an analyte of interest within the droplets according to various known methods.

Materials and Methods of the Invention [0028] Figure 1 illustrates an experimental setup for optical resonance experiments in microdroplets according to one embodiment. The main aspects of the set up include a droplet generator 1 10 (microchannel 1 10, discussed further below), two lasers 120, 190, a spectrometer 130, and a charge-coupled device (CCD) camera 140. [0029] The microchannel droplet generator 1 10 includes a T-junction 210 (e.g., see Figure 2A). The oil flows down the main channel 220 of the microchannel device 1 10. The diameter of the main channel 220 determines the diameter of the resulting droplets according to one embodiment. An aqueous liquid (containing fluorescent materials and possibly analytes of interest) flows out of a cross-channel 230 of the microchannel device 1 10. The aqueous liquid is sheared off by the oil at the T-junction 210 to form droplets of a size approximating the diameter of the main channel 220. The microchannel device 1 10 makes use of micro fabrication techniques according to one embodiment.

[0030] The droplets are first detected by a He-Ne laser 190 (Uniphase, Manteca, CA) and a photodiode 195 (Thorlabs, Newton, NJ). The red laser beam (633 run) is focused on the droplets at a closer vertical position to the T-junction and at a different angle than the Argon ion laser 120 and the forward-scattered light is collected through a series of lenses and filters 185 and focused onto the photodiode 195. The current induced in the photodiode is monitored through a digital oscilloscope 175 (LeCroy, WavePro 7000, Chestnut Ridge, NJ). The peak of the signal is set as a threshold for the droplet detection and used as a timer for triggering the delay generator 170. Two filters were used to avoid false signals due to the strong scattering from the Argon ion laser 120 and red fluorescence emission from Alexa Fluor 555. One of them is a shortwave cutoff filter at 540 run (Optosigma, Santa Ana, CA). This filter can block the scattered blue light from the laser. The second filter is a narrow-band interference filter (Optosigma, Santa Ana, CA), centered at 633 nm with a bandwidth of 1

nm. This filter blocks possible emission from Fluorescein or Alexa Fluor 555 dye in the droplet, which would interfere with the scattering light from the droplets through He-Ne laser 190. Since we observed the spectra in the 500-620 nm range, scattering from the droplets due to elastic scattering of He-Ne light did not affect our results. After the detection, the droplets were optically pumped by an Argon ion laser 120 (Coherent, Innova 70, Santa Clara, CA). The original beam (1.5 mm diameter at the 1/e ² point) was directed through a 488 nm bandpass filter (FWHM 1 nm, Optosigma, Santa Ana, CA) and was focused to a 50-micron spot to get better coupling of the pump beam into the microdroplets. The resultant fluorescent signal from the droplets was collected by a long working distance Piano Apochromat lens (Mitutoyo, Kawasaki, Japan); then focused through an objective lens (collection optics 150) onto a 150 mm focal length dual grating imaging spectrometer 130 (Acton Research Corporation, SpectraPro-150, Acton, MA). The scattered laser light collected by the collection lenses 150 was filtered by a holographic notch filter 160 (Kaiser Optical Systems Inc., Ann Arbor, MI) at 488 nm. The spectrometer 130 was connected to a TEK 512x512D front-illuminated thermoelectrically cooled CCD camera 140 (Princeton Instruments, Trenton, NJ). The data were collected and processed through the Princeton Instruments software controlling the camera shutter and the controller 180. The camera 140 and the droplet generator 1 10 were synchronized by the function generator and the delay generator 170 (Stanford Research Systems, DG535, Sunnyvale, CA). (0031] In the microchannel 1 10 including an immiscible medium, aqueous microdroplets that include glycerol and a fluorescent dye are formed according to one embodiment of the present invention. In one embodiment, the immiscible medium in the microchannel 1 10 is an oily medium, e.g., a low refractive index laser oil. The index of the oil is less than that of water according to one embodiment. In another embodiment, the refractive index of the oil is less than 1.33. The fluorescent material is selected from Rhodamine 6G, Fluorosene, or other organic dyes according to various embodiments. The concentration of glycerol is 50-80% by volume according to one embodiment. The glycerol concentration is 60-70% by volume according to another embodiment. The glycerol allows the droplet to achieve sufficient internal reflection for enabling MDRs. [0032] The oil flows down the main channel 220 of the microchannel device. Figure 2 A illustrates a microchannel 110 without droplets.. An aqueous liquid (containing fluorescent materials and possibly analytes of interest) flows out of a cross-channel 230 of the microchannel device 110. The aqueous liquid is sheared off by the oil at the T-junction 210 to

form slugs of water of a size approximating the diameter of the main channel 220. The channel expands to a larger size 240 in which the water slugs form droplets of diameters between 20 to 70 microns in diameter. The diameter is a determined by the relative flow rates of oil and water and is very reproducible (to within +/- 1 %). [0033] Figure 2B illustrates a microchannel 1 10 with droplets 250. In one embodiment, the droplets 250 are 20-70 microns in diameter. In another embodiment, the droplets 250 are 40- 60 microns in diameter. In yet another embodiment, the droplets250 are 50 microns in diameter. [0034] When the droplets 250 are excited by a suitable laser source, with sufficient energy and at the right wavelength to excite the fluorescence, MDRs are observed.

[0035] Figure 3 illustrates a laser beam 310 causing optical resonances 320 within a microdroplet 250 according to one embodiment. The intense optical field near the rim of the droplet 250 exhibits features that are characteristic of lasing. The optical field can be used to enhance normal fluorescence emissions and to enhance fluorescence resonance energy transfer. Because the optical field is confined to very near the rim of the droplet 250, only those fluorophores 330a within a threshold distance from the aqueous droplet/oily medium interface are excited efficiently, avoiding excitation of fluorophores 33Ob in the center of the droplet. The threshold distance is approximately one wavelength according to one embodiment. The threshold distance is 200 to 400 nm according to another embodiment. [0036] This selective excitation can be used to detect binding events of biological receptors that are immobilized at the oil/droplet interface with proteins or cells according to one embodiment. The microdroplet format can also be used with phosphor nanoparticles that are used for labeling biomolecules according to one embodiment. If a labeled molecule is trapped in a microdroplet in a channel, the particle can be excited continuously without photobleaching, enhancing the signal to noise ratio and without loss of the label by diffusion out of the measurement volume (of the microdroplet). This can be achieved by stopping the flow in the channel and trapping a single droplet in a laser beam. Because the spacing of the resonances of light in the droplet depends on the droplet diameter, which can in turn be determined by the design of the microchannel but most usefully by the relative flow rates of aqueous liquid and the oily medium, it is possible to tune an intense resonance to an absoφtion line of a compound of interest that might be present in a sample for example a trace pollutant. Scanning the resonance over the absorption line will allow sensitive

absoφtion measurements to be made by making use of the narrow line width of the resonance and the long path length of the high Q resonances in the microdroplets.

EXAMPLES [0037] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. [0038] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 ^rd Ed. (Plenum Press) VoIs A and B(1992).

Methods

[0039] We chose the avidin-biotin pair as described herein as a demonstration of FRET due to the high binding affinity between these molecules; this combination can be used efficiently for fiuoroimmunoassay applications in microdroplets. Avidin and biotin can be conjugated with Werent dye pairs using straightforward protocols and because of the relatively small size (biotin is a small molecule and avidin has dimensions of approximately 5.6 run x 5.0 nm x 4.0 nm); the distances are suitable to observe energy transfer between the dye pairs. One avidin has four biotin-binding sites. [0040] Initially, we studied the binding reaction between streptavidin and biotinylated BSA in a 96-well microtiter plate. Due to the considerable overlap of the emission spectrum of Rhodamine (absorption/emission maxima 525/547 nm) and absorption spectrum of Alexa Fluor 610 (absorption/emission maxima 612/628 nm) in the 550-600 nm range, these dyes can be used as a FRET pair to study the binding reaction between streptavidin and biotin. The

degrees of labeling for the conjugates used in this experiment were 5.1 Alexa Fluor 610 molecules per one streptavidin molecule and 1.3 Rhodamine molecules per one biotinylated BSA molecule (8 moles biotin/mol BSA). Solutions prepared in Phosphate Buffer Saline (PBS) solution were mixed in the 5-20 μg/ml range for both reagents and incubated for at least 30 minutes before data acquisition in a fluorescence plate reader. Measurements for solutions containing only BSA-biotin-Rhodamine and only streptavidin- Alexa Fluor 610 were performed for blank subtraction. The fluorescence intensity was measured between 550 nm and 700 nm (excitation: 525 nm) and the changes in the emission spectrum of Rhodamine and Alexa Fluor 610 were observed. After the blank subtraction, this binding reaction did not show any FRET. At constant Alexa Fluor 610 concentration, Rhodamine concentration was increased from 5 μg/ml to 20 μg/ml and no substantial change (less than 10%) in Alexa Fluor 610 emission was observed. To verify these data, the experiment was repeated with a nonspecific pair, replacing BSA-biotin-Rhodamine with BSA-Rhodamine (1.4 moles Rhodamine/mol BSA). Streptavidin- Alexa Fluor 610 and BSA-Rhodamine pair showed a similar spectrum with the biotinylated pair. This shows that avidin-biotin reaction did not result in FRET between the dyes. This could be due to the relatively long distance between donor and acceptor molecules due to the low donor concentration (1 ,3 Rhodamine molecule per BSA-biotin). [0041] Fluorescein-labeled biotin (biotin-4-fluorescein); streptavidin-Alexa Fluor 555 conjugate (3 moles dye/mol protein); streptavidin-Alexa Fluor 610 conjugate (5 moles dye/mol protein); biotin labeled FluoSpheres (40 nm, yellow/green fluorescent); neutravidin labeled FluoSpheres (40 nm, yellow/green fluorescent) and Alexa Fluor 555 dye were purchased from Molecular Probes (Eugene, OR). To prepare biotinylated BSA-Rhodamine conjugate, ImmunoPure biotinylated BSA (8 moles biotin/mol BSA) was purchased from Pierce (Rockford, IL). 5-carboxyrhodamine 6G, succinimidyl ester was obtained from Sigma (St Louis, MO). BSA-biotin and BSA were labeled with Rhodamine 6G following a standard procedure recommended by Molecular Probes. The degrees of labeling obtained were 1.3 moles RhodaminelmOl BSA-biotin and 1.4 moles/Rhodamine/mol BSA. FluoSpheres are polystyrene beads, which are internally loaded with a variety of dyes. Yellow-green fluorescent beads have excitation/emission maxima of 505/515 nm and thus are excited very efficiently using the 488 nm spectral line of the argon-ion laser. Biotin conjugated beads were used to observe the binding reaction between biotin and streptavidin. Neutravidin conjugated beads were used to conduct a control experiment for avidin-biotin reaction. Small bead size

(40 nm) was chosen for two reasons. First, to eliminate the background signal in the Alexa Fluor 555 emission range due a possible bulk fluorescence emission from the beads; second, to reduce the avidin binding ratio per bead and make it closer to the 4:1 ratio as observed in avidin-biotin binding in bulk solution. All solutions were prepared with PBS buffer (8.18 g/L NaCl, 2.98 g/L KCl, 0.27 g/L Na ₂ HPO ₄ JH ₂ O, 1.43 g/L KH ₂ PO ₄ , pH=7.5). After each run the microchannel was flushed with 20 ml deionized water (DI water) (Resistance = 18 MO) and operated for 5 minutes to avoid contamination between each run. A Spectramax M2 microplate reader (Molecular Devices, Sunnyvale, CA) and black 96-well plates from Nunc (Rochester, NY) were used for initial FRET studies.

Example 1: Effect of Inclusions on Microdroplet Resonances

[0042] Incubated and washed cell cultures (E-CoIi KB and Streptococcus) were diluted by 10 μM Rhodamine 6G in Phosphate Buffer Saline (PBS) solution for the experiments. The cultures were checked under a microscope for cell viability to assure that most of the cells were alive during the experimental run. The cell-Rhodamine 6G solution was used immediately after it was prepared to avoid any absorption/adsorption of fluorescent molecules by the cells which would reduce the concentration of free Rhodamine 6G in the solution. The cell concentration was checked in a 96-well plate reader with absorptivity measurements at 600 nm. Assigning an optical density of 1 at 600 nm to a concentration of 10 ⁸ cells/ml, we concluded that the cell cultures prepared had on the order of 10 ⁹ cells/ml. A calculation of the average number of cells per microdroplet followed from this cell concentration and the average microdroplet volume. With a cell concentration of 10 ⁹ cells/ml and an average droplet diameter of 20 microns, every microdroplet contained 0.3 cells on average. This means that ten microdroplets should yield observations of single cells in three cases. The MDR spectrum of 10 μM Rhodamine 6G in PBS solution (without cells) exhibits intense maxima at the first order modes of the microdroplet cavity.

[0043] Figure 4 shows the suppression of resonance peaks when the E-CoIi cells are added to the fluorescent solution. The sample solution comprises of 10 μM Rhodamine 6G in PBS with E CoIi cells (~10 ⁹ cells/ml). The sample size is a 20 micron droplet, which is about 4.18 pL.

[0044] When cells are added to the solution, the suppression of the resonance peak intensities are observed in the microdroplet spectrum. The effect of cell inclusions in the microdroplet is similar to quenching or a reduction in the Q factor of the cavity. This quenching effect can be

due to several causes: the local refractive index can change due to the presence of cell inside the microcavity; the cells can also cause scattering losses of light within the cavity. Although scattering can increase the mode visibility by increasing the photon leakage from the microcavity, it can cause reduction in Q factor due to asymmetry resulting in a reduction in resonance peak intensities. From the spectrum, one can observe that most of the first order modes are suppressed. Meanwhile, some of the higher order modes towards the blue end of the spectrum have higher intensities compared to the original spectrum. This is mainly because of the leakage of the high order modes due to scattering. Usually, the modes with higher mode-order number (1) have low intensities compared to the first order modes and tend to disappear first in quenching experiments. These results, in general, confirm previous experimental and theoretical studies on the effect of inclusions in microdroplets.

Example 2: Single Cell Inclusions in Microdroplets [0045] The intensity of the resonance peaks depends on the amount of fluorescence material inside the microcavity. Keeping the laser power constant, the main reason for reduction in intensity is the absorption or quenching which decreases the peak intensities through either radiative or non-radiative processes following excitation. In effect, the gain of the microdroplet cavity is reduced. There is no obvious spectral shift associated with the inclusion of cells into the optical cavity. The resonance peak locations remain unchanged. (0046] Figure 5 shows ten consecutive spectra from the cell detection experiments in microdrolets. Here, a single cell presence is detected in two droplets (spectra marked 510, 520) in a total often droplets observed through the suppression of optical resonance peaks. A single cell event is confirmed through the observation of a secondary dimmer diffraction pattern that is located in a position different from the primary diffraction pattern caused by the microdroplet alone. From the estimate that three out often microdroplets should contain a single bacterial cell, we expect that approximately three of the spectra should indicate the presence of a cell. Through the spectra, it can be seen that in two out often microdroplets, the resonance peak intensities have dropped dramatically. The number of cells in a single microdroplet should follow Poisson statistics, and it is possible to have more than one cell in each microdroplet when the resonance suppression is observed.

[0047] Single cell detection through optical resonance spectra was confirmed by the observation

of diffraction patterns through the interaction between the microdroplet and the laser focal spot. Whenever a single cell event is detected through the spectrum, a secondary dimmer diffraction pattern on top of the primary diffraction pattern is observed (not shown). This secondary diffraction appears at different locations on the primary one and it is caused by the interaction of between the laser light and the single bacterial cell in the microdroplet. This shows that the observation of resonant peak suppression in the microdroplet spectrum corresponds to a single cell detection event.

Example 3: Energy Transfer (FRET) |0048] To reduce the distance between the donor and the acceptor molecule, biotin-4- fluorescein (absorption/emission maxima 495/519 nm) and streptavidin conjugated with Alexa Fluor 555 (absorption/emission maxima 553/568 nm) pair was chosen thus excluding BSA from the reaction. The streptavidin-Alexa Fluor 555 concentration was kept in the 0-100 μg/ml range corresponding to 0-1.76 μM. The biotin-fluorescein concentration was varied between 1.4 and 1 1.2 μM to keep some excess fluorescein to account for the 4: 1 binding ratio. In this configuration, the donor molecule was fluorescein and the acceptor molecule was Alexa Fluor 555. Similar to the

Rhodamine/Alexa Fluor 610 pair, the Fluorescein/ Alexa Fluor 555 pair also has substantial overlap for their emission/absorption spectra. The labeling ratio was 3 Alexa Fluor 555 molecules per one streptavidin molecule and 1 fluorescein molecule for each biotin molecule. The fluorescence emission intensity measurements were performed in a microplate reader between 510 nm and 620 nm (excitation at 485 nm) following incubation for at least 30 min. After the blank subtraction, the emission spectrum for both dyes were compared. We observed a major decrease in fluorescein intensity while the streptavidin-Alexa Fluor 555 concentration was increased at a constant fluorescein-4- biotin concentration.

[0049] Figure 6 shows biotin-4-fluorescein (absorption/emission maxima 495/519 nm)and streptavidin-Alexa Fluor 555 (absorption/emission maxima 553/568 nm) interaction (emission spectra at 485 nm excitation). The spectra correspond to 5.6 biotin-4-fluorescein at different streptavidin-Alexa Fluor 555 concentrations (0-100 μg/ml). When the streptavidin- Alexa Fluor concentration was above saturation, all the biotin-fluorescein molecules in the solution were bound to streptavidin and fluorescein emission intensity dropped down dramatically. But this decrease in fluorescein emission did not result in an increase in Alexa Fluor 555 emission. This means that instead of a resonant energy transfer to Alexa Fluor 555,

the fluorescein emission was quenched by the binding reaction. Since the biotin-fluorescein complex is a small molecule (MW=644), the binding reaction caused static quenching by streptavidin. This was confirmed by a control experiment where biotin-fluorescein was mixed with avidin only. The same fluorescence intensity drop was observed. [0050] To avoid physical quenching due to binding, the link between biotin and the fluorescent tag should be sufficiently long. To achieve this, we used biotinylated fluorescent beads. As noted above, we used 40 nm biotinylated fluorescent polystyrene beads (absorption/emission maxima 505/515 nm) with streptavidin- Alexa Fluor 555 to observe FRET. The streptavidin-Alexa Fluor 555 concentration was kept in the range from 25-200 μg/ml, while the fluorescent bead concentration was changed between 0.0045% and 0.019% corresponding to 1.31-5.25 x 10 ¹² beads/ml. The fluorescent intensity measurements were performed at 500-620 nm (excitation at 485 nm) and emission peaks were observed with varying donor or acceptor concentration. The result was a decrease in fluorescent emission intensity from the nanobeads, corresponding to an increase in fluorescence intensity in Alexa Fluor 555 emission.

[0051] Figure 7 shows 40 nm biotinylated fluorescent polystyrene beads (absorption/emission maxima 505/515 nm) and streptavidin-Alexa Fluor 555 interaction (emission spectra at 485 nm excitation). The spectra presented correspond to 0.009% solid fluorescent beads and streptavidin-Alexa Fluor 555 at various concentrations (25-200 μg/ml). A decrease in fluorescent emission intensity from the nanobeads corresponding to an increase in fluorescence intensity in Alexa Fluor 555 emission was observed. This increase in acceptor intensity due to a decrease in donor emission implies an energy transfer between these molecules. [0052] This increase in acceptor intensity due to a decrease in donor emission implies an energy transfer between these molecules. Unlike the biotin-fluorescein and streptavidin- Alexa Fluor 555 reaction, the donor and acceptor molecules were close enough without being affected by the avidin-biotin interaction. We performed three control experiments to confirm this observation. Initially, to check whether observed intensity changes were due to specific binding, we replaced streptavidin-Alexa Fluor 555 with Alexa Fluor 555 only. As the Alexa Fluor 555 concentration was increased at constant fluorescent bead concentration, we did not neither observe a significant decrease in fluorescence emission from the beads nor an increase in Alexa Fluor 555 emission. Additionally, we substituted biotinylated fluorescent nanobeads with beads, which were conjugated to neutravidin. In this case, we did not expect

specific binding to occur between this pair. 0.1% BSA was added to the solution before incubation to reduce non-specific binding of streptavidin to the beads. As expected, fluorescence emission intensities revealed that there was no correlation between donor and acceptor emission intensities when there is no specific binding. As a final verification, we checked the reaction between the biotinylated fluorescent beads and avidin only. In this scheme, we observed whether the binding reaction affects the donor emission in the absence of acceptor molecule. The fluorescence intensity dropped less than (10% compared to 50% in FRET) compared to the case where the acceptor molecule was conjugated to avidin. This observed intensity drop for the fluorescent bead emission shows that part of the donor emission drop observed in streptavidin-Alexa Fluor 555 and biotinylated fluorescent bead reaction is due to the binding reaction itself and cannot be completely attributed to FRET between the labels.

Example 4; FRET in Microdroplets [0053] High optical intensities in microdroplets are able to increase the energy transfer efficiency for the FRET pairs, resulting in a high sensitivity biodetection scheme. To demonstrate this effect, we repeated the biotinylated fluorescent bead and streptavidin-Alexa Fluor 555 binding reaction in microdroplets using the setup shown in Figure 1. In a microdroplet, the emission from the fluorescent beads can be coupled to the resonances inside the cavity and increase the emission lifetime. This can result in much longer Forster radii than expected for the FRET pair. The same energy transfer pattern was achieved with microdroplets with a further decrease in donor emission fluorescence and a larger increase in acceptor emission because of improved energy transfer efficiency. [0054] Figure 8 shows biotinylated fluorescent bead and streptavidin-Alexa Fluor 555 binding reaction in microdroplets. The spectrum presented correspond to three different streptavidin-Alexa Fluor 555 concentrations (25, 100, 200 μg/ml) at constant fluorescent bead concentration (0.009% solids). As the streptavidin-Alexa Fluor 555 concentration is increased, there is a decrease in the donor (fluorescent bead) emission (500-550 nm; laser excitation at 488 nm). Morphology dependent resonances based on the fluorescence spectrum of fluorescent beads and Alexa Fluor 555 are also observed. In the microdroplet, the emission from the fluorescent beads can be coupled to the resonances inside the cavity and increase the emission fluorescence lifetime. This results in an enhanced energy transfer between donor and acceptor molecules.

[0055] Figure 9 shows acceptor (Streptavidin-Alexa Fluor 555) emission due to donor (biotinylated fluorescent bead) excitation through FRET as a result of the specific binding reaction in microdroplets. This figure shows the acceptor emission spectra in the 550-600 nm range and it is enlarged from Figure 8. [0056] Streptavidin-Alexa Fluor 555 concentration was kept in the 25-200 μg/ml range, while the fluorescent bead concentration was changed between 0.0045% and 0.019%. We observed the morphology dependent resonances based on the fluorescence spectrum of fluorescent beads and Alexa Fluor 555. We observed that there is some bulk fluorescence emission, which is not associated with the fluorescence resonance energy transfer. This is due to the fluorescent donor molecules located in the inner region of the beads, which are sufficiently removed from their acceptor pair to support FRET. As a result, the donor emission decreases but was not completely quenched because of the energy transfer. Reducing the bead size can reduce the bulk emission from the beads.

Additional Studies

[0057] To simulate the effect of cell presence on the optical resonances inside the microdroplets, we repeated the optical resonance experiments with microspheres. Two micron diameter silica and polystyrene sphere suspensions, Bangs Laboratories Inc. (Fishers, IN) were used. The results were surprisingly different in comparison with the cell solution. The presence of microspheres in the microdroplets did not suppress the optical resonances as the round- shaped cells did. The reason might be the complex optical characteristics of bacterial cells. The cells do not possess uniform optical properties like the silica and polystyrene microspheres. The average refractive index of a bacterial cell is roughly n = 1.3916 reflecting the different optical properties of its major components like the cell wall (n = 1.42) and the cytoplasm (n = 1.36). Simultaneous acquisition of the optical resonance spectrum and the elastic light scattering patterns might provide more detailed information about the optical properties, morphology and the location of the microparticle within the microdroplet; the additional information could provide some specificity to the measurement of individual bacteria or bioparticulates. [0058] We also examined the difference between the signal from the E. CoIi culture and the

Streptococcus culture to check the effect of inclusion morphology on resonance spectra. E. CoIi is rod-shaped and Streptococcus is round-shaped. Both types of cells are approximately 1-2 microns in size. The comparison of data from both experiments does not suggest an obvious difference in spectra that can differentiate between the shapes of the cells inside the microdroplet. Detailed analysis of higher order mode spectral peaks might reveal some information about the differences in the shape and size of the inclusion. It might also provide clues about the location of the inclusion inside the microdroplet. However, there is an indication that cell morphology can play a role in altering the shot-to-shot differences in resonance spectra. The round cells cause a constant reduction when the resonance suppression is observed; surprisingly, the rod-type cells show a variation in the spectra instead of a constant reduction in the intensity of resonances. The variation of intensity reduction arises from the random orientation of the rod cells with respect to the location of the resonances inside the spherical microcavity. Observations of this sort can be used as an indication of cell morphology. In addition, we checked the effect of cell viability on optical resonance spectra. To do this, the cell cultures were exposed to UV light for ten minutes. The cell viability was checked under a microscope. After UV exposure, the cell motility was reduced by more than 90%. When these cells were used for the experiment, the results showed no difference between the live cells and the dead ones. This is due to the fact that the cell membrane remains intact for a while after the exposure. In particular, E. CoIi cells have a cell wall in addition to the membrane that would maintain the same cell shape after lysis.

CONCLUSIONS

(0059] The detection of single bacterial cells in microdroplets has been demonstrated using optical resonances. At cell concentrations as low as one per microdroplet; the resonance peaks have been suppressed. The mechanism is mainly due to the local refractive index change and scattering caused by the cells. Absorption/adsorption of fluorescent molecules might have contributed further to the signal reduction. The detection system is non-specific; any inclusion that would cause a change in the optical characteristics of the microcavity would change the nature of the optical resonance peaks. The advantage of the system is the omission of the labeling procedure for the biomolecules to be detected. The fluorescent marker is simply added to the final solution without any specific binding procedure to the cells to be detected. The effect of the cell morphology and viability on the resonance peaks caused by

the fluorescent emission inside the microcavity was also studied. Cell morphology affected the variability of the spectra from droplet to droplet as rod-like cells rotated randomly inside the droplets. Cell viability did not affect the spectra. Further analysis of the data focused on the mode visibility and lasing thresholds might help differentiate different cell shapes and cell viability, especially when combined with additional scattering information.

[0060] Optical resonances in microdroplets were used to enhance the detection of binding reaction between avidin and biotin through FRET. Streptavidin and biotinylated BSA binding did not result in a non-radiative energy transfer between Rhodamine and Alexa Fluor 610. A better FRET pair selection and a higher labeling ratio (donor molecule per streptavidin) for the donor molecule may alter this conclusion. Static or contact quenching of biotin- fluorescein upon binding to avidin was observed. No significant increase in acceptor emission was observed during this interaction, which shows that the reduction in donor signal was not due to FRET but quenching. The use of a biotinylated fluorescent bead and strepmidin-Alexa Fluor 555 interaction caused a decrease in donor emission that was associated with an increase in acceptor emission. This energy transfer can be enhanced in a microdroplet because of the long photon interaction and residence time for high Q optical cavity modes. Since the fluorescence lifetime can be increased inside the microcavity and the coupled light can travel long distances around the droplet rim, a longer Forster radius is permissible and the detection limit can be improved. This detection method can be applied to a variety of immunoassay formats.

[0061] The location of the cell inside the microdroplet also affects the spectrum. The inclusion will have a greater effect if it is located at one of the focal points of the microdroplet when it is illuminated by the laser. In addition, the inclusion will affect the resonance peaks more if it is located within a wavelength of the rim of the microdroplet where the first order modes are strong. It is not possible to derive the position or the orientation of the cell in case of a rod-shaped cell from our data.

[0062] Some of the most important factors affecting the FRET effect due to specific binding are the number of fluorescent molecules conjugated to avididiotin; the ratio of the concentrations of labeled avidinhiotin; the temperature and pH of the media as it affects the fluorescence spectrum; the emissionlabsoqtion overlap of the donor/acceptor pair; and the excitation wavelength. The detection mechanism used in this work can be applied to several immunoassay formats, including a competitive fluorescence immunoassay. The antibody can be conjugated to the donor dye of a FRET pair while the antigen can be coupled to the

acceptor molecule. The initial emission from the acceptor molecule should be high because of the energy transfer between the donor and the acceptor. When an unknown amount of antigen is introduced into the solution, some of these antigens will replace the antigens labeled with the acceptor molecule and the change will be observed as a decrease in the acceptor emission. [0063] The microdroplet format, with its small volume and intense optical field, is ideal for binary detection of analytes. If a sample is diluted sufficiently so that the probability of more than one analyte molecule (a cell, a protein or DNA) is very small, single labels that correspond to single analyte molecules will be detected by the very evident change in the optical spectrum. Counting the number of droplets that exhibit a positive change will provide a measure of the concentration of analyte molecules in the original sample. Binary detection is very useful when significant background noise is present, as it is in biological samples. [0064] All references referred to herein are incorporated by reference in their entirety for all puφoses.

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