Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on

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A droplet-free digital enzyme-linked immunosorbent assay based on a tyramide signal amplification system Kenji Akama, Kentaro Shirai, and Seigo Suzuki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01148 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 20, 2016

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Analytical Chemistry

A droplet-free digital enzyme-linked immunosorbent assay based on a tyramide signal amplification system Kenji Akama*, Kentaro Shirai, Seigo Suzuki Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan E-mail: [email protected], Fax: 81-78-992-3284

ABSTRACT: Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horse radish peroxidase (HRP) labeled with an immunocomplex on beads and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freelymoving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Highly sensitive protein detection systems can be helpful when applied to early diagnostics and post-therapeutic monitoring. For example, hepatitis B virus (HBV) infection affects more than 350 million people worldwide and can cause acute or chronic liver disease; its management requires measurement of ultralow concentrations of hepatitis B surface antigen (HBsAg).1-5 However, the current standard protein detection method, the enzyme-linked immunosorbent assay (ELISA), is insufficiently sensitive to detect ultralow concentrations of protein biomarkers in a blood sample. Digital ELISA is a single molecule counting technology and one of the most sensitive immunoassay methods.6 In conventional ELISA, the limit of detection (LOD) is in the pM concentration range of the target protein, but Digital ELISA enables the detection of fM to aM concentrations. 7 For example, Kim et al. reported that the LOD of prostatespecific antigen was 2 aM, a dramatic improvement compared with conventional commercial ELISA (LOD = 3 pM, ADVIA Centaur, Siemens).8 The key point of Digital ELISA is the loading of each enzyme-labeled protein molecule individually into droplets in order to concentrate the enzyme reaction products in the droplets, thereby enabling detection of the signal from a single protein molecule. Digital ELISA requires a large number of femtoliter-sized drop-

lets, and several methods for generating the droplets have been proposed.9-12 Rissin et al. formed many droplets by etching tens of thousands of reaction vessels onto the end of an optical fiber bundle and sealing them.9 Sakakihara et al. generated droplets using a microchamber array formed on a hydrophilic-in-hydrophobic micropatterned surface.10 Over 106 droplets were formed simultaneously by injecting oil after the aqueous solution on the surface of the microchamber array. Using a different approach, Guan et al. reported the high-throughput formation of water-in-oil emulsions for single protein molecule detection using microfluidic channels.11 However, all these methods require specialized

Figure 1. Proposed principle of reaction of labeled tyramide substrate with a surface protein. HRP converts tyramide into tyramide radical, and the radical binds to aromatic moieties on the surface protein.

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Figure 2. Schematic illustration of droplet-free Digital ELISA based on a TSA system. (a) Single protein molecules are captured on beads and labeled with HRP. (b) Biotinylated tyramides react with HRP and are converted into tyramide radicals which then deposit onto beads with labeled HRP. (c) Streptavidin-labeled fluorescent dyes bind to biotin deposited on the beads. (d) The beads are counted digitally by flow cytometry. devices and accessories for preparing the droplets, and thus these measurement systems are rather large and complicated. Here, we propose a novel Digital ELISA method that does not require droplets. This droplet-free Digital ELISA utilizes the principles of tyramide signal amplification (TSA) using the catalyzed reporter deposition (CARD) technique.13-15 As shown in Figure 1, tyramide is a p-substituted phenolic compound, and horseradish peroxidase (HRP) can convert tyramide into a radical intermediate in the presence of hydrogen peroxide. These tyramide radicals form covalent bonds with aromatic compounds such as tyrosine and tryptophan on the surface of a protein. By pre-binding labeling molecules such as fluorescent dyes or biotins to tyramide, the labeling molecules can be deposited on proteins near the bound HRP. We conducted this reaction on freelymoving microbeads without using droplets: while dropletbased Digital ELISA enables the signal from a single target molecule to be detected by concentrating the enzyme reaction products in droplets, our method allows the detection of a similar signal by concentrating the enzyme reaction products on the surfaces of microbeads. Since no droplets are required, our method could realize a simple measurement system that does not require droplet-making devices and accessories such as pumps. Furthermore, since our approach does not require oil to make droplets, the sample is not contaminated with oil, and a conventional flow cytometer can be used for digital counting. This is important in terms of in vitro diagnosis because flow cytometry is widely used in clinical diagnosis due to its reliability and stability. 16-20 Droplet-free Digital ELISA using flow cytometry has potential as a next generation highly sensitive system for in vitro diagnostics.

tional Reference Standard) was purchased from the National Institute for Biological Standards and Control (United Kingdom). HBsAg negative serum from a healthy donor was purchased from Trina Bioreactives Ag. Washing buffer (HISCL washing solution) was obtained from Sysmex. Glass slides were purchased from Matsunami. Immunoassay. Capture antibodies were immobilized on the magnetic beads according to the manufacturer’s instructions, then the beads (3,000,000 beads) were dispersed in 1% BSA blocking buffer and incubated for 1 h at 25 °C with rotation. The beads were washed with washing buffer and dispersed in various concentrations of HBsAg solution (75 L) and incubated for 1 h at 25 °C with rotation. The beads were washed three times with washing buffer, then resuspended and incubated with a solution of biotinylated detection antibody (1.3 nM) for 1 h at 25 °C with rotation. The beads were washed three times with washing buffer, dispersed in streptavidin-labeled HRP solution (50 pM) for 1 h at 25 °C with rotation, then washed three times with washing buffer. As shown schematically in Figure 2 (a), this process provided a single target molecule and enzyme complex on the same bead. Enzyme reaction. The above beads were dispersed in biotinylated tyramide solution containing hydrogen peroxide, incubated for 30 min at 25 °C with rotation, then washed three times with washing buffer. Biotinylated tyramide was specifically deposited on beads labeled with HRP and not on non-labeled beads, as shown in Figure 2 (b). Fluorescent labeling. Bright fluorescent dyes are required to detect the signal from a single target molecule using flow cytometry. BV-421 is a synthetic fluorescent polymer that is dramatically brighter than other fluorescent dyes. 21 The above beads were resuspended in 1% streptavidin-labeled BV-421 solution and incubated for 30 min at 25 °C with rotation, then the beads were washed three times with washing buffer. As shown in Figure 2 (c), the biotin-labeled tyramide molecules deposited on the beads reacted with the streptavidin-labeled fluorescent dye, resulting in specific binding of the fluorescent dye to beads labeled with HRP. The use of BV-421-labeled tyramide rather than biotinylated tyramide and streptavidin-labeled BV-421 simplifies the labeling protocol. Detection of beads by microscopy. The fluorescentlylabeled beads were dispersed in PBS and placed on a clean

EXPERIMENTAL SECTION Materials. Magnetic beads 2.8 m in diameter were purchased from Thermo Fisher Scientific (Dynabeads M-270 epoxy). Anti-HBsAg antibody used as the capture antibody and biotinylated anti-HBsAg antibody used as the detection antibody were obtained from Sysmex. Streptavidin-labeled HRP and bovine serum albumin (BSA) were purchased from SDT. TSA kits were obtained from Perkin Elmer. BV421 streptavidin was purchased from BioLegend. Purified HBsAg (World Health Organization (WHO) Interna1


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Figure 3. (a)-(c) Overlay images of the fluorescence image and bright field image of beads with captured HBsAg at λ equal to 0.1 using a HRP-tyramide reaction time of 30 min. Bright spots on the beads are indicated by colored arrows in (a). Enlarged views of non-fluorescent beads and fluorescent beads are shown in (b) and (c), respectively. (d) Cross-sectional intensity line profile of the colored dashed line in (a). (4) [Ltotal ]=[L]+[AbL] Our approach requires an excess of beads over target biomarker molecules. When the target biomarker molecules react with capture antibodies on the bead, the number of biomarker molecules conjugated to the beads follows the Poisson distribution (eq. 5): (5) Pλk =(e-λ ×λk )/𝑘!

glass slide. The glass slide was mounted on a fluorescence microscope (BZ-X710, Keyence) equipped with a 40× objective lens (PlanApo NA0.95, Nikon), and fluorescence images and bright field images were acquired (excitation: 381–404 nm; emission: 417–477 nm; exposure time: 250 ms). The images were analyzed using ImageJ software (US National Institutes of Health).

λ=[AbL]/[beads]

Detection of beads by flow cytometry. The fluorescentlylabeled beads were dispersed in PBS and measured using flow cytometry (FACSVerse, BD Biosciences) as shown in Figure 2 (d). The flow cytometer was set up to detect the light intensity from forward scatter (FSC), side scatter (SSC), and fluorescence (excitation: 404 nm; emission: 425470 nm). The average flow velocity was 12 L/min. The flow cytometry data were analyzed using FlowJo software (FlowJo).

Pλk

where and λ represent the probability of conjugating k molecules to beads and the average number of target molecules on the beads, respectively. If λ is 0.1, the probabilities of 0, 1, or 2 molecules on the beads are expected to be 0.1 0.1 P0.1 0 = 90.5%, P1 = 9.0%, and P2 = 0.5% from eq. 5. In the measurement of HBsAg, where the number of beads is 3.0×106, the introduced target molecules (Ltotal) at λ equal to 0.1 is calculated to be 509 zmol using eqs. 2, 6, and the experimental values for [Abtotal] and KD of 7.8 nM and 0.16 nM, respectively (data not shown). The effect of labeling using the TSA method under the condition where most beads conjugate 0 or 1 target molecules was investigated. Beads captured HBsAg at λ equal to 0.1, then were labeled using the TSA method and observed under a fluorescence microscope. Two hundred and twenty fluorescence images and bright field images of different fields of view (FOV) were taken. The average bead number in an individual FOV was 576, and the total number of beads analyzed was over 120,000. Supplementary Figure 1 shows a typical FOV and Figure 3 (a) shows an enlarged view of a portion of a FOV. Most beads remained nonfluorescent (Figure 3 (b)) but several were fluorescent (Fig-

RESULTS AND DISCUSSION The reaction of target protein molecules (L) with antibodies on beads (Ab), resulting in the conjugation of the target protein molecules to the beads (AbL), can be predicted by an equilibrium equation (eq. 1).22 (1) Ab+L↔AbL Defining the dissociation constant (KD=[Ab][L]/[AbL]), it follows from eq. 1 that: (2) [AbL]2 -(KD +[Ltotal ])[AbL]+[Ltotal ][Abtotal ]=0 where [Abtotal] and [Ltotal] are given as eqs. 3-4: [Abtotal ]=[Ab]+[AbL]

(6)

(3)



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enzyme reaction conditions where the distance between beads is much larger than the diffusion length of tyramide radicals prevent tyramide radicals generated on one bead from depositing onto other beads. To maintain this condition, it is essential to mix well low concentrations of beads in the HRP-tyramide reaction. In this report, the bead concentration was adjusted to provide an average distance of 50 m between beads. Figure 3 indicates that the fluorescence signal is localized on the beads, and thus the diffusion length of the tyramide radicals is shorter than the diameter of the beads, allowing amplification of the signal without diffusion to other beads and without the use of droplets. Figure 4 shows histograms of the fluorescence intensity of beads captured with HBsAg at λ equal to 0.1 or 0, followed by various reaction times between HRP and the tyramide substrate (2 min, 10 min, 30 min). Fluorescence images and bright field images of different FOV were obtained by fluorescence microscopy, and over 700,000 beads were analyzed to determine the distribution of the fluorescence intensity of the beads. The depth of field of the objective lens (40×, NA0.95) was 0.6 m, which is smaller than the particle diameter of 2.8 m. It is therefore possible that the bright spot on a bead could be out of focus, depending on the position of the bright spot on a specific bead. In order to determine the fluorescence intensity of the bright spot accurately, only the center area of each bead was analyzed. Figure 4 shows that the fluorescence intensity of beads at λ equal to 0 remained 0 for 30 min, whereas the fluorescence intensity of some beads at λ equal to 0.1 increased and the peak of the histogram shifted to higher intensity values as the duration of the HRP-tyramide reaction increased. This increase in signal intensity indicates that the fluorescence signal is generated by the reaction between HRP and tyramide substrate. A reaction time of 30 min at 25 °C resulted in most positive beads having a much higher signal intensity than that of the negative beads. Supplementary Figure 2 shows the time course of the average number of tyramide molecules deposited on HRP-labeled bead. A reaction time of 30 min resulted in a saturating amount of deposited tyramide, estimated to be on average 670 tyramide molecules per HRP- labeled bead.

Figure 4. Time-course analysis of the HRP-tyramide reaction. The beads first captured HBsAg with λ equal to 0.1 (left column) or 0 (right column), then the beads were labeled with HRP, and finally HRP reacted with tyramide for 2, 10, or 30 min. ure 3 (c)). Figure 3 (d) shows the cross-sectional intensity line profile obtained from the colored dashed line in Figure 3 (a). The full width at half maximum (FWHM) in Figure 3 (d) was estimated to be about 0.65 ± 0.08 m (n = 30), which is less than the diameter of the beads, indicating that the fluorescence signal is localized on each bead. In dropletbased Digital ELISA, the droplet prevents the diffusion of fluorescent products from the droplet. Our approach does not utilize droplets, and thus it is possible that fluorescent products generated by one bead could diffuse and deposit onto other beads. However, the tyramide radicals generated by the HRP enzyme reaction are short-lived and thus their diffusion distance is limited to tens of nm. Therefore,

Figure 5. Droplet-free Digital ELISA based on a TSA system for HBsAg using flow cytometry. (a) A plot of forward scatter (FSC) and side scatter (SSC) from all beads. (b) A plot of SSC and fluorescence intensity of single beads (excitation: 404 nm; emission: 425-470 nm).


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Figure 6. Calibration curve for HBsAg spiked with human serum by droplet-free Digital ELISA based on a TSA system. The number of beads whose intensity was above the threshold were counted. Plots of the average enzyme per bead (AEB) versus the concentration of HBsAg are shown using (a) a log–log scale and (b) a linear–linear scale. The colored dashed line in (b) is the linear regression line. N = 5, and error bars represent 3 times the SD. The concentration of protein in the sample solutions was determined by counting the beads digitally using flow cytometry. Figure 5 shows the result of our Digital ELISA for HBsAg using human serum spiked with different concentrations of HBsAg. Figure 5 (a) shows a typical plot of the side scatter (SSC) and forward scatter (FSC) of the detected beads. The thresholds were set at 55,000 for both FSC and SSC in order to eliminate the effects of extraneous features such as debris, then the signal over the threshold was detected. Digital ELISA requires counting the number of single beads. We identified single beads from a plot of SSC versus FSC and counted them. Over 95% of the beads were detected as single beads and the others were detected as doublets and higher order aggregates. Since most beads in the sample were detected using flow cytometry, the singlebead detection efficiency was approximately 95%, a much higher value than the 50% detection efficiency of introduced beads in a previous study using a femtoliter droplet array.23 Supplementary Figure 3 shows typical plots of SSC and fluorescence intensity of all detected beads for the different concentrations of HBsAg tested, and Figure 5 (b) shows a plot of SSC and fluorescence intensity of single beads defined in Figure 5 (a). The population of beads with high fluorescence intensity increased in an HBsAg concentration-dependent manner. However, it was challenging to completely separate target positive beads and target negative beads because their populations overlapped. This overlap could result from the deposition of tyramide being localized on the beads and because the beads were observed only from one direction by the flow cytometer, as shown in Figure 2 (d). Since these beads are composed of iron oxide, they are not transparent to visible light. So, depending on the angle between the bright spots on the beads and the flow cytometer detector, fluorescent light from a bead could be blocked by the bead itself, resulting in decreased detected fluorescence intensity. It should be possible to address this

problem by detecting the beads from more than one direction using a multi-detector. We analyzed the flow cytometry data and generated a calibration curve for HBsAg (Figure 6). Based on the fluorescence intensity of the target negative beads, the threshold was set as the average intensity plus 5 times the standard deviation (SD), as shown in Figure 5 (b). We identified the beads whose fluorescence intensity was above the threshold as target positive beads. The y-axis of Figure 6 shows the average number of enzyme molecules per bead (AEB) determined from the fraction of bright beads. 24 At low concentrations of HBsAg, when AEB is less than 0.7, the HBsAg concentration was quantified by digital counting. At high concentrations of HBsAg, when AEB is greater than 0.7, the HBsAg concentration was quantified by the fluorescence intensity of all the detected beads.24 HBsAg is used in clinical applications for screening HBV, therefore both a low LOD and a wide dynamic range are needed.2 Our experimental results were lower than the theoretical values determined by eqs. 2 and 5, as shown in Figure 6 (a), but the results nonetheless demonstrate a dynamic range of over 4 orders of magnitude, which is useful for screening applications. The LOD was estimated by defining the HBsAg concentration at an average signal equal to the average blank signal plus 3 times the SD. As shown in Figure 6 (b), the lowest concentration of HBsAg detected was 0.25 mIU/mL (375 aM), and the LOD was calculated to be 0.09 mIU/mL (139 aM) from the linear regression. Therefore, when applied to HBsAg detection, our approach achieved >20-fold higher sensitivity than conventional ELISA (LOD of 5 mIU/mL, or 7.5 fM).25 In our method, 0.09% of the detected beads were false positives, and this blank noise was mainly due to non-specific binding of HRP on the target negative beads, as is also the case with droplet-based Digital ELISA.6,8 The effect of non-specific binding could be suppressed by improving the blocking conditions and washing



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lower than 1.0 (0.68), indicating that the detection efficiency of our approach is lower than that of droplet-based Digital ELISA. The low detection efficiency may be due to difficulty in detecting the localized fluorescence signal, depending on the angle between the bright spots on the beads and the detector. This could be improved by detection with a multi-detector, as mentioned above.

CONCLUSIONS We have developed a droplet-free Digital ELISA using a TSA system. HBsAg is a clinical biomarker and its detection using our Digital ELISA correlated significantly with a droplet-based Digital ELISA. Furthermore, our approach showed a wide dynamic range of over 4 orders of magnitude and an improved LOD of 0.09 mIU/mL (139 aM), a >20-fold higher sensitivity than conventional ELISA. These results could be useful for the management of HBV. Our approach permits the highly sensitive detection of protein biomarkers without the need for equipment to make waterin-oil droplets, such as microfluidic channels or array devices, and thus our method has potential for minimizing the measurement system and avoiding contamination of the sample with oil. Further, our method utilizes conventional flow cytometers currently used clinically. Taken together, these are critical advantages for use in in vitro diagnostics. We believe that this technique holds promise for highly sensitive diagnostic systems for the early detection, monitoring, or treatment of disease.

Figure 7. Comparison between droplet-free Digital ELISA and a droplet-based Digital ELISA. The same sample comprising beads with captured HBsAg and labeled HRP were measured by each method. method, or by using an immunocomplex transfer (ICT) method. The ICT method involves the formation of immunocomplexes of enzyme-labeled products of the antigenantibody reaction on beads, as shown in Figure 2 (a). In this step, some of the enzyme molecules are absorbed on the surface of beads non-specifically. In order to avoid the effect of these non-specific binding molecules, only immunocomplexes are disassociated from the surface of the beads. The dissociated immunocomplexes are subsequently captured by 2nd clean beads, then enzyme molecules on the 2nd beads react with a substrate. This approach can suppresses the blank noise that non-specific binding enzyme molecules cause.26,27 The application of ICT mothod to our method would reduce the noise due to non-specific binding components and thus likely improve its sensitivity. Figure 7 shows a comparison of our approach with a droplet-based Digital ELISA. The minimum reproducibly detectable signal was set as the calibration point, and the relative values of signals to this calibration point were plotted for different concentrations of HBsAg. HBsAg concentrations ranging from 0.2 to 25 mIU/mL in human serum (n = 24) were captured on beads and labeled by HRP, as shown in Figure 2 (a), then the beads were divided into two groups and detected by each method. Droplet-based Digital ELISA utilized a femtoliter droplet array, as reported by Kim et al. 8 10-Acetyl-3,7-dihydroxyphenoxazine (QuantaRed enhanced chemifluorescent HRP substrate, Thermo Fisher Scientific) was used as the fluorescent HRP substrate and is a more sensitive substrate than Amplex Red, the fluorescent substrate previously used for single molecule detection using a femtoliter droplet array.28,29 Supplementary Figure 4 shows representative bright field and fluorescence images of femtoliter droplet array beads loaded with QuantaRed at different concentrations of HBsAg. Over 80,000 beads in the images were analyzed to obtain the calibration curve shown in Supplementary Figure 5. The results indicate that it is possible to obtain concentration-dependent signals from HBsAg using the femtoliter droplet array and QuantaRed substrate system. According to Figure 7, our approach correlated significantly with a droplet-based Digital ELISA (r2 = 0.98). However, the slope of the linear regression fit was

ASSOCIATED CONTENT Supporting Information. Additional information noted in the text. Available free of charge on the ACS Publication website at http://pubs.acs.org.

Conflict of Interesting Disclosure. The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Fax: 81-78-992-3284

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