A bead-based immunogold-silver staining assay on capillary-driven microfluidics
Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in “bead lanes”, which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using “classical” enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL−1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL−1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.
KeywordsMicrofluidics Silver staining Immunoassays Microbeads
Healthcare facilities in the developing world consist not only of centralised laboratories in well-equipped hospitals in cities, but also of primary healthcare centres with limited infrastructure in peripheral regions. In such settings, POC diagnostics are critical for guiding appropriate treatments for infectious diseases such as malaria, in which the delay between diagnosis and treatment can be life threatening (Peeling and Mabey 2010). Developments of innovative technologies has transformed POC diagnostics to bring healthcare services closer to patients (BIO Ventures for Global Health 2010). There are many benefits that well reflect this transformation, for example the increasing number of rapid antigen detecting tests, or POC immunodiagnostic assays for malaria and HIV (UNITAID 2017, 2016). There are two main diagnostic platforms that are being used for POC applications: conventional lateral flow assay technology and emerging microfluidics-based technologies (Sharma et al. 2015). While the lateral flow technology has a long history with a number of successful stories in commercialising diagnostic products, there are only very few microfluidics-based tests available in the market (Chin et al. 2012).
Sensitive detection and simplicity of use are prerequisites for POC diagnostic assays (Yager et al. 2006). In other words, compelling performance and affordability are current challenges for microfluidics-based diagnostic chips (Chin et al. 2012). Fluorescence, luminescence and absorbance are three optical detection methods that have been implemented for signal transduction of the immunobinding reactions in low cost, microfluidics-based POC diagnostics (Baker et al. 2009; Gai et al. 2011; Kuswandi et al. 2007). Fluorescence-based assays offer high sensitivity and selectivity (Lin et al. 2011). Such immunoassays rely on fluorophores that are linked to detection antibodies (dAb) and that can emit a fluorescence signal upon excitation using a specific wavelength. The intensity of the fluorescence signal is proportional to the amount of antigens selectively captured on a surface. Organic fluorescent dyes and quantum dots are commonly used as labels. For example, an ultrasensitive fluorescence immunoassay was developed to simultaneously detect two cancer biomarkers, carcinoma embryonic antigen and α-fetoprotein, in serum using CdTe/Cds quantum dots as fluorescent probes (Hu et al. 2010). The limit of detection (LOD) of this fluorescence immunoassay on the microfluidic chips was 250 femtomolar, which is 3 orders of magnitude better than that of assays using conventional fluorescence probes. Issues that are hindering further progress on fluorescence-based POC diagnostics are the (photo)stability of fluorophores and the cost, complexity and fragility of fluorescence readers. Luminescence is a method used in variants of standard immunosorbent assays. In luminescence immunoassays, an enzyme conjugated to the dAb converts a substrate into a product that emits light (Mirasoli et al. 2014). Unlike fluorescence, this method does not require an optical excitation source, therefore luminescence detection is simpler than that of fluorescence in terms of optics. In a recent example showing the implementation of this technique in a portable and microfluidics-based diagnostic platform, a chemiluminescent assay detecting C-reactive protein (CRP), a biomarker that indicates inflammation in the body when its concentration exceeds 5 μg mL−1, was developed (Hu et al. 2017). In this prototype, the LOD for CRP reached 4.27 ng mL−1, which is comparable to conventional chemiluminescent immunoassays in laboratories. Furthermore, the compact design of this prototype with all-integrated reagents and pre-programmed on-chip mechanical valves for controlling the steps of the assay showed the technical feasibility of implementing a luminescence assay into a POC platform. Nevertheless, the stability and cost of the enzyme used for such assays need to be addressed. Hydrogen peroxide or chemicals that are needed for chemiluminescence are unstable at ambient conditions and require storage at low temperatures, which is also cumbersome for the development of fully integrated POC diagnostic devices.
Perhaps the most popular detection method used in commercially-available diagnostic immunoassays is the one based on absorbance detection. Absorbance-based assays rely on the conversion of a substrate into a strongly coloured product by an enzyme, which is linked to a dAb (Novo et al. 2011). Although being slightly less sensitive than fluorescence- and luminescence-based immunoassays, these assays offer a reasonable performance and can involve relatively low cost reagents and chemicals (Shekarchi et al. 1985; Yu et al. 2011). To improve the performance of assays using absorbance measurements, metal nanoparticles (NPs) such as gold NPs are used to intensify colorimetric signals of assays (Xu et al. 2009). Typically, the size of gold NPs varies from 10 to 50 nm (Sun et al. 2014). Larger gold NPs might create steric hindrance for ligand-receptor interactions and smaller gold NPs can be challenging to visualize (Liu et al. 2014). Holgate et al. proposed to use immunogold silver staining (IGSS) to solve this problem by depositing silver on the surface of gold NPs to enhance the signal intensity after the ligand-receptor binding has taken place (Holgate et al. 1983). To investigate the suitability of IGSS for diagnostic assays, a lateral flow assay for influenza was engineered, detecting hemagglutinin of H5-type of influenza viruses (Wada et al. 2011). LOD of the silver amplified assay was decreased 500 times compared to that of the assay without silver amplification, and this LOD was 10 times lower than that of commercial influenza rapid diagnostic tests. Similarly, the IGSS method was used to detect HIV and rubella infections in blood samples and the LODs were comparable to the reference methods (Patel et al. 1991; Rocks et al. 1991). The specificity of both assays using IGSS was also higher and interferences due to cellular components of whole blood specimens were less prominent than with other commonly used diagnostic methods.
In this paper, we present a capillary-driven microfluidic chip for IGSS with capture antibodies (cAbs) for analytes located on the surface of microbeads. We specifically developed an assay using species and anti-species polyclonal antibodies to demonstrate how such assay can be implemented. There are three key features in this approach. First, the beads are self-assembled from a bead suspension into specific structures of the chip and the chip is sealed by lamination with a dry film resist (DFR) layer at low temperature. This makes the integration of beads and biological receptors into the microfluidic chip simple, fast and versatile. Second, the core of the beads is fluorescent and the growth of a metallic silver layer during the silver staining step efficiently attenuates/quenches this fluorescence, which also eliminates the need for fully transparent microfluidic devices for measuring absorption of light by the formed silver film. The fluorescence signals are extremely strong when no analyte is present in the sample and fluorescent beads are highly stable against photobleaching. Third, the microfluidic chip is designed to accommodate sub-microliters of solutions, which are sequentially pipetted for the IGSS assay. All handling steps for the assay take as little as 20 min and there is no need for active microfluidic elements to perform the assay.
2 Materials and methods
2.1 Antibodies and reagents
All reagents were purchased from Sigma-Aldrich unless otherwise stated. Water was purified using a Simplicity 185 system (Millipore, Billerica, MA). Phosphate buffered saline (PBS) solution was prepared by dissolving commercially available PBS tablets. A solution of 1% w/v bovine serum albumin (BSA) was prepared in PBS. PBS with Tween 20 (PBST) was prepared by adding Tween 20 to PBS to a resulting concentration of 1%. 3,3′-5,5′-tetramethylbenzidine in water (BM Blue POD substrate) was used as a substrate for enzyme-based immunoassays. A 1:1 v/v mixture of silver A and silver B from SE 100–1 KT silver enhancer kit was prepared prior to the signal amplification step. Streptavidin-coated 96-well microtiter plates and blocker BSA solution were from Life Technologies. Fluorescent streptavidin PMMA beads (PolyAn GmbH, Red 25, 5.9 μm diameter) were used as carriers to integrate the cAb in microfluidic chips. Biotinylated anti-rabbit IgGs was used as cAb. Donkey anti-rabbit IgGs conjugated with 6 nm gold nanoparticles (abcam) and donkey anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (abcam) were used as dAbs. Rabbit IgGs and mouse IgGs were used for positive and negative control experiments for immunoassays on 96-well microtiter plates.
2.2 Fabrication materials
Si wafer (Si-Mat, Kaufering, Germany) and SU-8 photoresist (SU-8 3010, MicroChem Corp.) were used to fabricate microfluidic chips. Acetone and isopropyl alcohol were used to clean the microfluidic chips after dicing. DFR (DF-1050, Engineered Materials Systems, Inc., USA) was used to seal the chip.
2.3 Protocols for assays
To validate the reagents and characterize the assay protocol, the IGSS assay was first performed using standard streptavidin-coated 96-well microtiter plates. Between each step, the plates were washed three times using a plate washer (Tecan). First, 50 μL of a biotinylated anti-rabbit IgG solution (10 μg mL−1) was added into each well and incubated for 30 min at room temperature (RT). Then solutions of rabbit IgG (positive control) and mouse IgG (negative control), were added into each well using a serial dilution factor of 2 with the starting concentration 4 μg mL−1 in the first wells (50 μL/well). The incubation was 30 min at RT. For silver staining, a solution of gold conjugate was diluted 1:2000 (v/v) in 1% BSA in PBST and added into each well (50 μL/well). Incubation of the analyte with dAb-conjugated gold was 15 min at RT. Solutions of silver A and silver B were mixed (1:1, v/v) and added immediately into each well (50 μL/well). The silver development step was 20 min, at RT and in the dark (plates covered with an aluminium foil). For standard enzymatic assays, a solution of HRP conjugate was diluted 1:10′000 (v/v) in 1% BSA in PBST and added into each well (50 μL/well). This incubation was 60 min at RT. Solutions containing POD blue were added (50 μL/well) and the enzymatic reaction was allowed to proceed for 20 min. To measure the end-point absorbance, a plate reader (Sunrise, Tecan) was used at a wavelength of 570 nm and the absorbance measurements were taken at RT.
For immunoassays performed on microfluidic chips, aliquots of solutions were sequentially added to the loading pads of the chip. Fluorescence images of the beads integrated into the microfluidic chip before and after the silver staining step were taken using a fluorescence microscope (Nikon Eclipse 90i, Japan) equipped with a 20× objective and a Texas Red fluorescence filter. Excitation of fluorophores was done using an LED Lumencor lamp (software SOLA S2 Controller). Images were taken using a black and white CCD camera (DS-1 QM, Nikon) and an acquisition time of 400 ms and using a ND16 filter. The software Fiji (ImageJ) was used to analyse the fluorescence images. Fluorescence images for regions of interest (ROI) comprising the beads before and after silver staining were acquired and the mean fluorescence intensity for each experiment was obtained by subtracting the background signal around an ROI to the mean signal value in the ROI.
3 Results and discussion
3.1 Assay and detection principles
The first added liquid is a 1% solution of BSA in PBS and it is allowed to flow for 1.5 min (i.e. ~53 nL based on optically monitoring the advancement of the solution in the chip) to block surfaces and prevent non-specific adsorption of analytes and dAbs up to the receptor areas. The assay proceeds with the addition of the pre-mixed antigen-dAb solution (1:1 v/v), which is allowed to flow for 5 min (~175 nL). The antigen-dAb complex is captured by the cAb present on the beads during this step, Fig. 1b. Then, deionised water is added for a 4-min rinsing step (~140 nL) before adding a silver staining solution, which is allowed to flow for 7 min (~246 nL). In presence of antigens, a film of metallic silver forms on the surface of beads. During this silver staining step, the microfluidic chip is covered with an aluminium foil to avoid non-specific, light-catalysed reduction of silver. The addition of deionised water displaces the silver staining solution where beads are located and stops the formation of the silver film so that the fluorescence emitted from beads can be measured.
3.2 Chip design and bead integration
The bead integration step is initially facilitated by capillary actions followed by evaporation of carrier solution, Fig. 3b. 0.5 μL bead solution in 1% BSA in PBST (0.01%) (0.5% w/v) is introduced at the bead loading pad when the chip surface is still open. Bead solution flows orthogonal to the main channel and only beads are trapped at the bead lane. More bead solution can be loaded if needed until the bead lane is completely filled with beads. Lateral spreading of the excess liquid in the main channel is minimized owing to the lower capillary pressure in the main channel compared to the bead lanes. Because the beads self-assembly process happens without using any external energy, many bead lanes can be filled with beads in a short time by serial deposition of the carrier solution. This makes the technique compatible with mass production using automated pin spotters. Following the integration of the beads, the chip surface needs to be sealed to create a strong capillary pressure of samples required for the assay and to minimize evaporation. Many sealing techniques are available for microfluidic applications, such as adhesive or thermal bonding. However, these techniques are typically not compatible with integrated reagents because of the use of solvents and/or high temperatures. Here, the sealing of the chip is performed by gently laminating a 50-μm-thick DFR at 45 °C. This low temperature and fast (<10 s) sealing process ensures proteins coated on the bead surface are minimally affected, Fig. 3c. In addition, it is not required to structure the DFR to pattern the openings for the loading pads. A straight cut aligned to the loading pads is sufficient to hold the pipetted liquid owing to the anti-wetting structures. Because all channel walls are inherently hydrophilic, no additional surface treatment is needed for the capillary flow. Finally, ready-to-use chips can be singulated by manual breaking through the partially diced regions, Fig. 3d (Temiz and Delamarche 2014). This step also breaks the DFR, making the whole process compatible with wafer-level reagent integration and sealing. For a standard clean room using 200 mm Si wafers, this process would yield more than 300 chips per wafer.
3.3 Assay implementation validation
To characterize the growth of the silver film over time, the IGSS assay was performed using a sample containing a rabbit IgG at a concentration of 4 μg mL−1 on microtiter plates and the silver staining process was stopped using three minute intervals. Absorbance measurements taken using a bench-top plate reader are presented in Fig. 5b. The growth of the silver film can be divided into three phases: first, the silver growth exhibits a slow initial phase for approximately 9 min, then the silver staining evolves rapidly between 9 to 21 min. During this time, the thickness of the silver film is proportional to the duration of the silver reduction reaction. After 21 min, the silver staining process reaches plateau, which is typical of electroless plating reactions and in agreement with previously reported data (Hayat 1995).
A microfluidic chip with successful implementation of silver staining and complementary integration of microbeads was developed. The use of fluorescent beads for microfluidics-based immunodiagnostic assays is not a limitation for point-of-care applications because many fluorescence devices have been engineered and fluorescence readers have been developed for various point-of-care applications. The assay with silver staining on chip achieved comparable sensitivity when compared with microtiter plates assays in less than 20 min. Moreover, the implementation of immunogold silver staining in microfluidics system eliminates the need for fully transparent devices when absorption of light is measured, and allows a simple in-plane optical detection system. Effect of temperatures on performance of assays was investigated, presenting no adverse effect for the silver staining immunoassays. While shelf life of reagents and their stability was not investigated in this work, this should not cause any issues since antibodies, commercially-available silver staining solutions and reagents are stable and commonly used for both research and in vitro diagnostic applications. The proof-of-concept prototype developed in this work achieves similar performance to that of lab-based immunoassays while retaining most of the advantages of rapid tests. The time taken to carry out an assay is only 20 min and there is no need for active microfluidic elements. Once the liquid is introduced, the flow is self-driven. The chips are designed to be compact and simple to use. Furthermore, the potential for mass production is considered by using dry film resist for easy fabrication, and by utilizing a design on Si that can easily be transferred to available mass production technologies. Possibly, this system can be fully integrated with automatic microfluidic delivery of buffers and reagents. Overall, we demonstrated strategies with which microfluidic chips and microbeads can be combined with silver staining to achieve a performance comparable to a reference lab-based method.
Ngoc M. Pham is supported through the Engineering for Development doctoral scholarship by ETH Global and the Sawiris Foundation for Social Development. Walter Karlen is supported through the Swiss National Science Foundation professorship award 150640 “Intelligent point-of-care monitoring”. Yuksel Temiz, Robert D. Lovchik and Emmanuel Delamarche thank Elisa Hemmig and Onur Gökçe for discussions and Walter Riess and the IBM Research Frontiers Institute for their continuous support.
- BIO Ventures for Global Health, The Diagnostics Innovation Map: Medical Diagnostics for the Unmet Needs of the Developing World (2010)Google Scholar
- M. A. Hayat (ed.), Immunogold-Silver Staining: Principles, Methods, and Applications (CRC Press, Taylor & Francis Group, 1995)Google Scholar
- C. Holgate, P. Jackson, P. Cowen, C. Bird, J. Histochem. Cytochem. 31(7), 938-944 (1983)Google Scholar
- H. Gai, Y. Li, E. S. Yeung, Top Curr Chem 304, 171-201 (2011)Google Scholar
- I.C. Shekarchi, J.L. Sever, L. Nerurkar, D. Fuccillo, J. Clin. Microbiol. 21, 92 (1985)Google Scholar
- UNITAID, Malaria Diagnostics Technology and Market Landscape (2016), https://unitaid.eu/assets/Malaria_Diagnostics_Technology_and_Market_Landscape_3rd_Edition_April_2016-1.pdf. Accessed 14 May 2018
- UNITAID, HIV Rapid Diagnostics for Self-Testing, 3rd edn. (2017), https://unitaid.eu/assets/HIV-Rapid-Diagnostic-Tests-for-Self-Testing_Landscape-Report_3rd-edition_July-2017.pdf. Accessed 14 May 2018