Droplet Sensitized Fluorescence Detection for Enzyme-Linked

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Droplet Sensitized Fluorescence Detection for EnzymeLinked Immune Sorbent Assays on Microwell Plate Tsuguhiro Kaneko, Yue Sun, Hizuru Nakajima, Katsumi Uchiyama, and Hulie Zeng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05668 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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Analytical Chemistry

Droplet Sensitized Fluorescence Detection for Enzyme-Linked Immune Sorbent Assays on Microwell Plate Tsuguhiro Kaneko‡, Yue Sun†, Hizuru Nakajima‡, Katsumi Uchiyama‡ , Hulie Zeng† ‡ * † School

of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China

‡

Department of Applied Chemistry for Environment, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan * Corresponding author: Tel/Fax: +86-21-51980056, Email: [email protected]

ABSTRACT: Microwell plate/microtiter plate is among the most widely used tools for use in immune assays. In this paper, we report on a sensitive method for enhancing fluorescence emission detection by simply adding several droplets of an immiscible organic compound into the microwells before detection. To prove the concept, human IgA was determined on a microwell plate using this droplet enhanced fluorescence (DEF) detection method. An obvious enhancement in fluorescence was observed. The detection limit (LOD) was about 1/20 times and the sensitivity was 4 times greater than that without droplets. To prove the use of the method for disease diagnosis, the IgG of measles in human plasma was determined using the proposed DEF method. A LOD of around 1/5 times and a sensitivity of 4 times for the DEF were easily achieved compared to ELISA with a conventional fluorescence detection. INTRODUCTION The microwell plate assay system was invented on the 1950’s and it has since evolved to be a popular tool for a wide range of bio-measurements. Benefiting from the mechanism of highly selective and specific solid-liquid affinity reaction on the microwells, measurements made on a microwell plate showed a high accuracy and a high precision. The microwell plate has been adopted to not only the well-known enzymelinked immune sorbent assay (ELISA) based on a typical affinity reaction between an antigen and an antibody but also to the measurement of pathogens1, estimation of cytotoxicity2, sorting cells3, monitoring the molecular recognition 4 and to the determination of the reaction constant of inhibitors 5-7. Relying on the other outstanding features of microwell plate in high throughput measurements, it also has been used to screen for drugs, inhibitors and their inducers 8-11. In fact, the most popular application of the microwell plate is still on ELISA for bio-measurements, and the method has now been commercialized 12-14. Although the microwell plate has been applied to various fields in bio-analysis, early detection nearly always involves UV-Vis absorption measurements 15-17, which has the obvious drawbacks of a low sensitivity and a high limit of detection (LOD). The microwell plate has been recognized as a classic tool that can be used in numerous areas of bio-analysis and is now considered to be the standard method18-21. Because the microwell plate is so widely used, the ELISA technique has enjoyed numerous applications. Numerous attempts have been made to decrease the consumption of reagents and sediment, to enhance the sensitivity of the method and to lower the LOD 22-24. Regarding the research to improve the sensitivity, the

development of highly sensitive detection methods or imaging methods to achieve a high resolution and a high sensitivity for microwell plate has been a subject of interest in recent years2226. For instance, a photonic crystal biosensor has been coupled with a microwell plate to develop a sensitive lable-free detection method 22. Polyelectrolyte (PEL) layers deposited with silver or gold nanoparticles 23, pure sliver or polymer layer 24 have been modified on the inner well of microwells to enhance fluorescence emission; the adoption of protein induced fluorescence enhancement (PIFE) has been developed to obtain a higher sensitivity for fluorescence detection in microwell plate 25. In addition, fluorescence imaging has also been developed of the use in conjunction with a microwell plate to achieve high-throughput quantitative imaging 26. Within the above areas of research, fluorescence emission detection is generally considered to be the most sensitive method and has attracted the attention of numerous researchers. One way to improve the sensitivity of fluorescence detection is to improve the efficiency of collection of fluorescence emission by the design of devices. For example, the three dimensional microlens array 27-29 and micro-reflectors 30 both have been developed to effectively increase the collection efficiency of fluorescence emission. The other way to improve fluorescence detection involves the production of strong electromagnetic field at the solid-liquid interface that can efficiently excite the ambient fluorophores to improve fluorescence emission efficiency 31, 32. However, a practical method to enhance fluorescence emission and with the capability to directly enhance the fluorescence detection for microwell plate is still not available.

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Based on the previous droplet enhance fluorescence (DEF) technique developed in our group 33, the sensitivity of fluorescence detection could be improved via a droplet microresonator. Both the exciting light and the emission ray could be multi-reflected within the droplet microresonator, and the efficiency of light excitation and the fluorescence collection would be increased. Similarly, we found that fluorescence detection of microwell plate can be sensitized by the addition of the droplets microresonators into the microwells. The only step is simply adding benzyl benzoate (BnBzO) droplets into the microwells before detection. By doing this, the sensitivity of fluorescence detection could be greatly enhanced, resulting in a lower LOD than that for the conventional method. The enhancement in the fluorescence detection of the microwell plate is experimentally confirmed in this report.

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the ejected BnBzo and measured by a fluorescence microscope. The fluorescence emission of the resorufin solution and the BnBzO droplet was observed by a fluorescence microscope. The intensity of the fluorescence of BnBzO droplet and the background of resorufin solution were simulated by means of the Origin 8 program. Optimizing the Number of BnBzO Droplets for the Standard Microwell Plate Based on the above-determined optimized size of the BnBzO droplet, different numbers of optimum sized BnBzO droplets was injected into the microwell that had firstly been loaded with 20 μL of a 5 μM resorufin solution. The microwell plate was then set into the microwell plate reader to detect the fluorescence signal.

MATERIALS AND METHODS Materials and Apparatus Benzyl benzoate (BnBzO) and Resorufin were purchased from Sigma-Aldrich (Missouri, USA). A human IgA kit, including affinity purified goat anti-human IgA, a human reference serum solution and a goat anti-human IgA HRP conjugated, was purchased from Bethyl Laboratories (Montgomery, TX, USA). Measles immunoglobulin G (Ig G) was obtained from the Hue University Hospital (Hue, Vietnam). Bovine serum albumin (BSA) was obtained from Merck (Calbiochem, Darmstadt, Germany). 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent) was purchased from Life Technologies (Invitrogen, Eugene, OR, USA). It was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 13.8 mmol/L for use as a storage solution and was stored in the refrigerator at -20 °C prior to use. The working substrate solution of Amplex® Red was prepared by mixing with phosphate-buffered saline (PBS) buffer (pH=7.4) and H2O2 solution just before use. Na2HPO4, NaH2PO4 and H2O2 were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Washing buffer (PBST) was prepared with 0.5% (w/v) BSA/0.1% (v/v) Tween-20 (Kanto Chemical Co. Inc., Tokyo, Japan) in 100 mmol/L PBS (pH = 7.4). All solutions used in the experiments were prepared by ultra-pure water purified by Milli-Q pure water system (Tokyo, Japan). A fluorescence microscope (Olympus, Tokyo, Japan) with a U-FGW filter set was used to observe the fluorescence image of the droplet and a 96-well microwell plate reader (Tecon Spectra Fluor, MTXLab Systems, Inc., Florida, USA) was utilized to observe/collect the fluorescence signal of the 96well microwell plate. Optimizing the BnBzO Droplet Size In order to obtain the optimal droplet size needed to enhance fluorescence detection, different-sized BnBzO droplets were generated using a micro-syringe/micro-pipette and were injected into a microwell containing a 5 μM resorufin solution. The diameter of the droplet can be calculated by the volume of

Figure 1. Procedure to perform ELISA coupling with DEF sensitized fluorescence detection, the only step was done for DEF detection was highlighted by a line square.

ELISA Fluorescence Measurement Performed by a Microscope and by a Microwell Plate Reader The measurement of human IgA was performed on a 96-well microwell plate following the standard ELISA protocol with an additional procedure for adding BnBzo droplets at the last step, as shown in Figure 1. Briefly, 30 μL of a 10 μg/mL goat anti-human IgA solution was introduced into a 96-well plate, and incubated for coating at 4 °C overnight. After washing three times with 60 μL of PBST buffer, 60 μL of BSA (1%, w/v) blocking buffer was introduced into each microwell which were then incubated for 1 h at room temperature. After washing the plate three times with 60 μL of PBST buffer, 30 μL of a human IgA solution at different concentrations were introduced into the microwells and the plate was incubated for 1 h at room temperature. Each concentration of human IgA was replicated four times. The plates were then washed with PBST buffer three times, and 30 μL of a 2.5 μg/mL HRP labeled goat anti-human Ig A solution was added to each microwell. After a 1 h incubation at room temperature, the microwells were washed with PBST buffer and 30 μL of Amplex® Red substrate was then added to each microwell to generate fluorescent resorufin. The BnBzO droplets were introduced into each microwell in the last step and the plate was set under an inverse type incident light fluorescence


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microscope and set into the microwell plate reader to detect the fluorescence signals, respectively. Diagnosis of Measles by measuring Ig G by DEF Coupled Microwell Plate Similar to the measurement of human Ig A by ELISA coupled with DEF, the measurement of the measles Ig G was proceeded as the standard ELISA procedure with BnBzo droplets being added just before the fluorescence detection, which was observed by the fluorescence microscope and by the microwell plate reader, respectively. RESULTS AND DISCUSSION

the size of the BnBzO droplet can also have a significant effect on the efficiency of the DEF. In the section, we report on introducing different sized BnBzO droplets, and observations of the fluorescence under a fluorescence microscope. As shown in Figure 3a, the intensity of the fluorescence of the BnBzO droplet in 5 μM Resorufin was increased with increasing diameter of the BnBzO droplet until the diameter of the droplet reached 700 μm. When the diameter of BnBzO droplet was larger than 700 μm, the BnBzO droplet began to lose its spherical shape, shown in Figure S1, and the enhancement in fluorescence via the droplet micro-resonator would be lost. Therefore, a BnBzO droplet with a diameter of 700 μm was selected as the optimum size for use in the following experiments.

Principles of DEF Based on previous research, we confirmed the DEF phenomena when an immiscible organic droplet was induced into the aqueous solution. Both the exciting radiation from the light source and the fluorescence emission of the ambient fluorophores would be enhanced at the interface of a transparent droplet 34. The efficiency to the fluorescence emission detection of the microwell plate would be greatly improved when transparent BnBzO droplets are added at the last step when the droplet phase has a higher refractive index than that of the ambient aqueous solution, as shown in Figure 2 and Table S1. Actually, the generation of fluorescence enhancement by a spherical droplet microresonator only needs an ambient fluorescent liquid with a depth of several nanometers 35. Thus, the amount of fluorescent liquid required for DEF is low, indicating that DEF has great potential for detecting sensitive emission in small sample sizes. Theoretically, all of the emission detection, i.e., chemiluminescence and fluorescence can be enhanced by the proposed DEF method for the multi-reflection inside the micro-spherical droplet with a higher refractive index within the droplet, the enhancement in fluorescence detection could be magnified via both the multi-reflection of the exciting light and the multi-reflection of the emission ray, thus producing a highly efficient excitation rate and a sufficient collection rate. Significantly, the only step for the proposed method was the addition of the simple injection of organic droplets into the microwells at the last step. This would constitute an easy and acceptable method for the general operator to have a sensitive fluorescence detection or chemiluminescence emission detection. Exploring the Optimum Size and Number of BnBzO droplets needed Usually, microresonators with micro-sphere, micro-toroid and micro-disk shapes would have a high efficiency in terms of enhancing the multi-reflection inside 36, therefore the shape of the droplet would affect how efficient the enhanced fluorescence will be. We confirmed that the optimal shape of the BnBzo droplet was within 1000 μm, as shown in Figure S1. Although a transparent BnBzO droplet with a high refractive index could improve the efficiency of fluorescence detection,

Figure 2. The use of DEF for a microwell plate, the upper amplified picture shows the microwell accepting BnBzo droplets and an enhanced fluorescence was obtained from the higher refractive index of droplet phase.

In fact, the total fluorescence intensity detected was increased with increasing number of BnBzO droplets based on observations with a microwell plate reader, as shown in Figure 3b. When the number of 700 μm o.d. BnBzO droplets reached 15, the enhancement in fluorescence detection in the microwell reached the optimum value. Once the number of the BnBzO droplets exceeded to 15 in the standard microwell, the BnBzO droplets trended to fuse together and to change their size and lose the shape of a sphere at a diameter of 700 μm. Therefore, the DEF effect by the BnBzO droplets was



weakened when more than 15 BnBzO droplets were used, as shown in Figure 3b. In summary, 15 BnBzO droplets with diameters of 700 μm were optimal for a standard 7 mm of microwell to give the maximum enhancement in fluorescence detection when the microwell plate reader was adopted as the detector. Performance of ELISA by DEF on Microwell Plate To confirm the possible application of DEF in fluorescence detection for microwell plate, the measurement of human IgA was performed by the standard procedure and BnBzO droplets with diameters of 700 μm were injected into each microwell at the end of last step. Then, the fluorescence detection was respectively performed by the fluorescence microscope and the microwell plate reader, and the calibration curves for human IgA obtained by the microscope are shown in Figure 4 and the data were listed in Table 1.

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Obvious enhancements in fluorescence were achieved in the detections via the fluorescence microscope and microwell plate reader as shown in Table 1. The DEF detection observed by the fluorescence microscope showed a nearly 4 times larger sensitivity and 1/20 of the LOD compared with that of a conventional fluorescence detection. In addition, the DEF signal collected by the fluorescence microscope appeared to have a higher sensitivity than that when the microwell plate reader was used. The LOD for DEF observed by the fluorescence microscope and by the microwell plate reader were both decreased, the LOD for DEF obtained by the fluorescence microscope was lower than that by the microwell plate reader in Table 1.

Figure 4. Calibration curve of human IgA in the sensitized fluorescence detection by DEF observed by a fluorescence microscope.

Figure 3. Optimizing the size of the BnBzo droplets in a 5 μM resorufin solution in DEF in a microwell plate (a) and Optimizing the number of the BnBzo droplets in a 5 μM resorufin solution in DEF in a microwell plate (b).

Based on the above findings, we conclude that the DEF detection observed by the fluorescence microscope or by the microwell plate reader were both enhanced. However, the use of the fluorescence microscope resulted in a higher sensitivity compared to the fluorescence collected by the microwell plate reader. One reason for this is that the observation of fluorescence imaging obtained by the fluorescence microscope was focused on a planer transverse section of the droplet, as shown in Figure S2. The fluorescence signal observed by the fluorescence microscope reflected the fluorescence intensity of each droplet, as a result, the detected fluorescence for the droplet was more intense. While, the fluorescence detection carried by the microwell plate reader collected the total fluorescence emission from the top of the microwell plate. It should be noted that the fluorescence signal of droplets obtained by this way needed to be illuminated through the solution layer to reach the fluorescence detection window. Because the density of the BnBzo droplet was larger than that of water, the BnBzo droplets were located at the bottom of the microwells, as shown in Table S1. Therefore, the fluorescence signal collected by the microwell plate reader was more mild compare with that observed by a fluorescence microscope. Application of DEF to the Diagnosis of Measles


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To confirm the practical application of the proposed DEF, we carried out the detection of measles Ig G utilizing the microwell plate coupled with DEF. The measles IgG could be sensitively detected by ELISA coupled with DEF, and the sensitivity of the measurement could be enhanced by DEF using either the fluorescence microscope or the microwell plate reader as detectors, as shown in Figure 5 and Table 2. Actually, a larger sensitivity could be achieved when a fluorescence microscope is used. Using the microwell plate reader, a 4 times higher sensitivity was found, and a 4 times lower LOD was found for DEF compared with the traditional fluorescent solution detection. Table 1. Calibration curves of human IgA observed by the fluorescence microscope and the microwell plate reader.

Measurement of human IgA with the added BnBzo droplets by microscope Measruement of human IgA by microscope Measurement of human IgA with the added BnBzO droplets by microwell reader Measurement of human IgA with traditional ELISA

Linear relationship*

R2

LOD(ng/mL)

Y=129.44X-16.761

0.9921

0.129

Y=35.821X+4.1578

Y=592.37X+2686.8

Y=549.67X+2890.9

0.9980

2.948

0.9854

0.210

0.9858

0.588

* The linear relationships were performed from 0 to 30 ng/mL.

larger detection sensitivity observed by the fluorescence microscope was obtained because of the focus on the planer transverse section of the droplet resulted in the detection of a larger fluorescence intensity as the shown in Figure S3. CONCLUSION Fluorescence emission in a microwell plate could be improved by adding droplets of an immiscible organic compound to the microwells, resulting in a larger refractive index than that of the ambient solution. Experimentally, the sensitivity and the LOD of the fluorescence detection were both improved by simply adding droplets of an immiscible organic solvent at the end of last step in the ELISA measurement. The DEF for microwell plate was experimentally confirmed by the measurement of IgA. And the practicability of the DEF for microwell plate was demonstrated by the measurement of Ig G of measles. We conclude that the DEF method could be extended to all types of emission detections, including chemiluminescence detection. Table 2. Calibration curves of measles IgG observed by a fluorescence microscope and a microwell plate reader. Linear relationship*

R2

LOD(ng/mL)

Measurement of measles IgG with the added BnBzo droplet by microscope

Y=134.02X+301.7

0.9845

0.240

Measurement measles IgG microscope

Y=36.491X+17.037

0.9940

0.594

Measurement of mealses IgG with the added BnBzO droplets by microwell reader

Y=836.07X+5712.8

0.9465

0.351

Measurement of mealses IgG with traditional ELISA

Y=708.43X+5649.4

0.9423

1.209

of by

* The linear relationships were performed from 0 to 30 ng/mL.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge via the ACS Publications website at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Figure 5. Calibration curves of measles IgG obtained by a fluorescence microscope.

We conclude that the simple addition of BnBzo droplets to microwells could improve the sensitivity of fluorescence detection. Similar to the above detection of human Ig A, a

* H. Zeng, [email protected] Notes The authors declare no competing financial interest.



ACKNOWLEDGMENT The authors acknowledge the financial support to Dr. H. Zeng from the Japan Society for the Promotion of Science (JSPS) 18K05178 and from the Starting Grant of Fudan University. REFERENCES (1) Gamble, R.; Muriana, P. M. Appl. Envir. Microbio. 2007, 76(13), 5235-5244. (2) Frgala, T.; Kalous, O.; Proffitt, R. T.; Patrick Reynolds, C. Mol. Cancer Ther. 2007, 6(3), 886-897. (3) Stagoj, M. N.; Komel, R.; Comino, A. Bio. Techniq. 2004, 36(3), 380-382. (4) Wentland, M. P.; Raza, S.; Gao, Y. J. Chem. Edu. 2004, 81(3), 398-400. (5) Lobmaier, C.; Hawa, G.; Go tzinger, M.; Wirth, M.; Pittner, F.; Gabor, F. J. Mol. Recognit. 2001, 14, 215-222. (6) Li, S.; Li, R; Dong, M.; Zhang, L.; Jiang, Y.; Chen, L.; Qi, W.; Wang, H. Sensor. Actuat: B. Chem., 2016, 222, 198-204. (7) Liu, H.; Feng, L.; Cai, Y.; Hua, Y.; Liu, M.; Yin, M.; Li, S.; Lv, X.; Wen, J.; Wang, H. J. Mater. Chem. B, 2018, 6, 75037510. (8) Reid, B. G.; Stratton, M. S.; Bowers, S.; Cavasin, M. A.; Demos-Davies, K. M.; Susano, I.; McKinsey, T. A. J. Mol. Cell. Cardio. 2016, 97, 106-113. (9) Bader, C.; Chelladurai, J. J.; CindyHall, K. T.; ,Carlson, S. A.; Brewer, M. T. Vet. Parasito. 2016, 223, 34-37. (10) Shi, X.; Sha, S.; Liu, L.; Li, X.; Ma, Y. Anal. Biochem. 2016, 498, 53-58 (11) Derbré, S.; Gatto, J.; Pelleray, A.; Coulon, L.; Séraphin, D.; Richomme, P. Anal. Bioanal. Chem. 2010, 398,1747-1758. (12) www.gbo.com Microplate Selection Guide; 2016. (13) www.bosterbio.com ELISA Handbook; (14) Crowther, J. R. The ELISA Guidebook,2nd ed.; Springer: Germany 2009. (15) Simonney, N.; Labrousse, H.; Ternynck, T.; Lagrange, P. H. J. Immunolo. Meth. 1996, 199, 101-105. (16) Zhang, Q., Zhang, J.; Shen, J.; Silva, A.; Dennis, D. A.; Barrow, C. J. J. Appl. Phycol. 2006, 18, 445-450. (17) Cho, Y. A.; Kim, Y. J.; Hammock, B. D.; Lee, Y. T.; Lee, H.-S.; J. Agric. Food Chem. 2003, 51 (27), 7854-7860. (18) Lai, S.; Wang, S.; Luo, J.; James Lee, L.; Yang, S.-T.; Madou, M. J. Anal. Chem. 2004, 76, 1832-1837. (19) Morais, S.; Tortajada-Genaro, L. A.; Arnandis-Chover, T.; Puchades, R.; Maquieira, A. Anal. Chem. 2009, 81, 56465654. (20) Liu, W.; Chen, D.; Du, W.; Nichols, K. P.; Ismagilov, R. F. Anal. Chem. 2010, 82, 3276-3282. (21) Sista, R.; Hua, Z.; Thwar, P.; Sudarsan, A.; Srinivasan, V.; Eckhardt, A.; Pollack M.; Pamula, V. Lab Chip 2008, 8, 2091-2104. (22) Choi C. J.; Cunningham, B. T. Lab Chip 2007, 7, 1-8. (23) Nooney, R. I.; Stranik, O.; McDonagh, C.; MacCraith, B. D. Langmuir 2008, 24, 11261-11267. (24) Yano, K.; Iwasaki, A. Sensors 2017, 17, 37(10p). (25) Valuchova, S.; Fulnecek, J.; Petrov, A. P.; Tripsianes, K.; Riha, K. Sci. Rep. 2016, 6, 39653(10p). (26) Durai, A. P.; Sankaran, K.; Muttan, S. EJBI 2013, 9(2), 5868. (27) Yang, H.; Gijs, M. A. M. Anal. Chem., 2013, 85 (4), 20642071 (28) Fan, C.; Lv, X.; Liu, F.; Feng, L.; Liu, M.; Cai, Y.; Liu, H.;

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Figure 1. Procedure to perform ELISA coupling with DEF sensitized fluorescence detection, the only step was done for DEF detection was highlighted by a line square. 88x100mm (300 x 300 DPI)



Figure 2. The use of DEF for a microwell plate, the upper amplified picture shows the microwell accepting BnBzo droplets and an enhanced fluorescence was obtained from the higher refractive index of droplet phase. 141x73mm (300 x 300 DPI)


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Figure 3. Optimizing the size of the BnBzo droplets in a 5 μM resorufin solution in DEF in a microwell plate (a) and Optimizing the number of the BnBzo droplets in a 5 μM resorufin solution in DEF in a microwell plate (b).



Figure 4. Calibration curve of human IgA in the sensitized fluorescence detection by DEF observed by a fluorescence microscope.


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Figure 5. Calibration curves of measles IgG obtained by a fluorescence microscope.


Droplet Sensitized Fluorescence Detection for Enzyme-Linked

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