Yeast surface display-based microfluidic immunoassay.

In this paper, we present a new microfluidic immunoassay platform, which is based on the synergistic combination of the yeast surface display (YSD) technique and the microfluidic technology. Utilizing the YSD technique, antigens specific to the target antibody are displayed on the surface of engineered yeast cells with intracellular fluorescent proteins. The displayed antigens are then used for the detection of the target antibody, with the yeast cells as fluorescent labels. Multiplex immunoassay can be readily realized by using yeast cells expressing different intracellular fluorescent proteins to display different antigens. The implementation of this YSD-based immunoassay on the microfluidic platform eliminates the need for the bulky, complex and expensive flow cytometer. To improve the detection sensitivity and to eliminate the need for pumping, a functionalized micro pillar array (MPA) is incorporated in the microfluidic chip, resulting in a detection limit of 5 ng/mL (or 1 ng in terms of amount) and enhanced compatibility with practical applications such as clinical biopsy. This new platform has a high potential to be integrated into microfluidic detection systems to enable portable diagnostics in the future.


Introduction
Fast and highly sensitive immunoassay plays an important role in medical and life sciences. In particular, the analysis of specific antibodies in human serum with high specificity and sensitivity is becoming an increasingly critical task as the process enables early diagnosis of many diseases, including infectious diseases such as HIV and hepatitis B [1], autoimmune diseases [2] such as systemic lupus erythematosus (SLE) [3,4], as well as allergies and cancers [5][6][7]. Traditional immunoassay technologies are mostly based on fluorescence and enzyme activities, such as enzyme linked immunosorbent assay (ELISA) and fluorescent immunoassay (FIA), which have well-documented protocols and good sensitivity and specificity. However, these immunoassays involve time consuming processes and can only be performed in centralized laboratories.
Recently, there is an urgent global need for immunoassay platforms that are compatible with portable applications, in particular, point-of-care testing. Microfluidic immunoassays are innovative technologies with the potential to realize point-of-care diagnostics, integrating analytical procedures such as sample preparation, separation, reaction and detection on a single chip. Based on these technologies, portability, simple operation and short assay time can be achieved with less sample consumption. For example, derived from DNA microarrays, peptide microarrays that feature a large number of protein probes at discrete locations within a small area have become increasingly accessible and more widely applied in simultaneous analysis of a large number of target analytes, facilitating serodiagnostics of various diseases in numerous groups [8][9][10]. Moreover, Chen et al. incorporated all nucleocapsid protein fragments in a protein microarray to study the antigenicity of different regions of severe acute respiratory syndrome (SARS) coronavirus to fight against SARS [11]. Luminex assays are another multiplexed immunoassay platform recently developed based on xMAP technology (multi-analyte profiling), which enables simultaneous detection and quantification of multiple targets [12]. Multiplexing of up to 100 unique assays within one single sample was realized by the xMAP system which combines flow cytometry with fluorescent-dyed microbeads, lasers, and digital signal processing [13][14][15][16]. In the heterogeneous immunoassay, the key to constructing a microchip for effective separation of labeled and unlabeled analyte are the stable immobilization of specific biological active molecules. A number of different surface modification and covalent conjugation methods have been developed [17,18], including physical adsorption [19,20], direct chemical covalent conjugation [18,21], spacer-added chemical covalent conjugation [22,23], and biological affinity interactions [24][25][26]. However, most of the available techniques require tedious steps of antigen purification and wet-chemistry processes to immobilize the probe antigen. Besides, it may be difficult for the target antibody to approach the antigen conjugated to a solid phase, resulting in reduced detection sensitivity. Moreover, the time consuming and laborious antigen purification and labeling processes significantly reduce the appeal and limit the application of immunoassay in antibody-based diagnostics.
Yeast surface display (YSD), first reported in 1997, is a molecular display system, which, by using engineered yeast cells, can display analyte-specific bio-moieties such as antigens or antibodies on the cell surface. YSD enables easy production of purified antigen/antibody by simply yeast culture and centrifugation, eliminating the tedious steps of traditional antigen/antibody purification. Our group developed a yeast surface display-based cell counting immunoassay (YSD-CCI), which, with the help of a flow cytometer, enabled determination of the target antibody quantity by counting the yeast cells attached with the antibody analyte instead of measuring enzyme activity or fluorescence intensity [27]. This method provides higher sensitive than yeast-ELISA, which employs enzyme labeled antibody for signal development, and is compatible with multiplexed antibody detection. However, the bulky, complex and expensive fluorescent flow cytometer is required, which undermines the application potential of this method, especially in scenarios where resources are limited or portability is needed.
In this study, in order to render the application of the YSD-CCI method in resource-limited or portability-required situations, we implemented it on a microfluidic platform. A prototypical microfluidic immunoassay device was developed, which substituted the flow cytometer with a functionalized microfluidic chip. Multiplex antibody detection was realized using bi-functional engineered yeast cells. A functionalized micro pillar array (MPA) was fabricated and incorporated in the microfluidic chip to achieve higher detection sensitivity and to replace pumping with onchip capillary-driven liquid flow. This microfluidics-based on-chip YSD-CCI method is believed to have excellent compatibility with applications such as clinical biopsy used in general hospitals, as well as high potential to be integrated in portable antibody-based molecular diagnostic systems.

Materials
The yeast strain EBY100 and display vector pCT were obtained from the laboratory of Dr. K. Dane Wittrup (Massachusetts Institute of Technology) and Dr. Eric V. Shusta (University of Wisconsin-Madison), respectively. The yeast expression vector YEP 181 was provided by Dr. Mingyong Xiong of HKUST. The anti-c-myc antibody, anti-HA antibody and HRP conjugated goat-anti-mouse antibody were purchased from Santa Cruz Biotechnology, Inc. 3-Aminopropyl trimethoxysilane (APTMS), poly-l-lysine, Albumin from bovine serum (BSA) and glutaraldehyde were purchased in Sigma-Aldrich. Protein G was bought from Shanghai Raygene Biotechnology. Other chemicals employed were of analytical reagent grade.

Chip fabrication and functionalization
The PDMS-glass microfluidic device was constructed by simple one-mask soft lithography and molding. A channel with a length of 15,000 m and a width of 4500 m was fabricated on the PDMS, and the glass surface in the channel was functionalized with protein G through covalent conjugation. Briefly, the surface was modified with 5% (v/v) APTMS in ethanol for 2 h with the coupling agent 5% (v/v) glutaraldehyde. Next, the chip was treated with 5 mg/mL poly-l-lysine as the space linker for 6 h. After that, another 2 h glutaraldehyde treatment was applied. Finally, protein G at a concentration of 2 mg/mL in phosphate buffered solution (PBS) was allowed to react with the modified glass surface for 6 h before blocking with 4% BSA. The microfluidic chip incorporating a MPA was constructed on a silicon wafer by deep reactive ion etching (DRIE). Considering the size of the yeast cells, we designed an equilateral triangular arrangement of a micro pillar array with a 30 m distance between the pillars and a 15 m shift after every 3 rows, which had been demonstrated to be the most efficient geometry for cell capturing [28]. Computational analysis of the flow distribution and cell trajectories performed using the commercially available finite element microfluidics solver COMSOL determined that the flow rate in our experiment was 0.18 L/s, while the linear flow rate was 100 m/s.

Cell culture
The bi-functional yeast cells YEP-M-HA (displaying HA tag on the surface and expressing intracellular mCherry) and YEP-G-c-myc (displaying c-myc tag and expressing enhanced green fluorescent protein, EGFP) were constructed by our group previously, and were grown in SDD medium (yeast synthetic dropout medium with dextrose) at 30 • C (in a 250 rpm orbital shaker) for 24 h. The Leu − and Trp − dropout mediums were used to select yeast cells expressing yeast expression vector YEP 181 and yeast display vector pCT, respectively. To induce the display of HA or cmyc tag, cells from the SDD cultures (of OD 600 between 2 and 5) were collected to inoculate 3 mL of SDG medium (yeast synthetic dropout medium with galactose) to a starting OD 600 of about 0.5. These cultures were grown at 30 • C. It was estimated that each cell displays approximately 10 4 HA or c-myc antigens after induction with galactose [29].

YSD-based microfluidic immunoassay on the PDMS-glass chip
In the YSD-based microfluidic immunoassay performed on the PDMS-glass chip, 200 L of 2 × 10 7 CFU/mL YEP-M-HA was separated from the culture medium by centrifugation and incubated in 4% BSA in PBS to block the yeast surface. The blocking by BSA ensures that only the target antibodies will bind to the yeast surface through interaction with the displayed specific antigens, so that non-specific binding of non-target antibodies to the yeast surface can be minimized. After washing with wash buffer (PBS with 0.4% BSA), 200 L of anti-HA antibody at varying concentrations (from 5 ng/mL to 5000 ng/mL) was applied and incubated for 1 h. A sample without anti-HA was used as negative control. After washing, the yeast cell suspension was introduced into the protein G-modified microchannel. The samples were injected at 2 L/min with a syringe pump. After that, fluidic force discrimination (FFD) [30], which utilizes microfluidic force to preferentially remove nonspecifically bound cells. The density of cells remaining after FFD was obtained by optical counting and was used to determine the target antibody concentration. A series of images of the capture zone was collected utilizing a CCD camera and a fluorescent microscope, Nikon TE2000-U. Using NIH (National Institutes of Health) recommended image processing software, Image J, the number of cells within the microscope field (4.754 mm 2 ) is determined by setting a binary threshold to delineate the cells.

Multiplexed YSD-based microfluidic immunoassay on the PDMS-glass chip
200 L of 2 × 10 7 CFU/mL YEP-M-HA and YEP-G-c-myc yeast cells were mixed at 1:1 ratio. A sample with 1000 ng/mL anti-cmyc antibodies and 1000 ng/mL anti-HA antibodies served as the target, while a sample without any antibody was used as negative control. FFD was performed with PBS buffer as described above.

YSD-based microfluidic immunoassay on the MPA silicon chip
In the YSD-based microfluidic immunoassay performed on the MPA silicon chip, 2 × 10 7 CFU/mL YEP-G-c-myc was used and incubated with a series of 10-fold dilutions (from 5000 ng/mL to 0.5 ng/mL) of the target, anti-c-myc antibody. A sample without anti-c-myc was used as negative control. After washing by centrifugation with PBS, 20 L of the yeast suspension was pipetted onto the protein G-modified MPA-incorporated PDMS-silicon chip, which had been blocked with 4% BSA previously. An absorbent wicking was fixed at the output region of the chip to keep the flow continuously. A washing step was performed by pipetting 200 L of 0.4% BSA in PBS onto the entire area of the chip. After that, the washing buffer was removed by pipette and wicking. After washing for several times, the yeast cells captured on the chip were counted by an upright microscope.

YSD-based microfluidic immunoassay on the PDMS-glass chip
The working principle of the YSD-based microfluidic immunoassay on the PDMS-glass chip is illustrated in Fig. 1. Antibody-specific epitopes are displayed on the yeast cell wall, while fluorescent proteins are expressed inside the cell. Affinity interaction between the target antibody and the epitope produces analyte-specific "labeled" yeast cell conjugates, which are then introduced into a protein G-modified microfluidic device. Vertical hydrodynamic focusing (HDF) [31,32] is used to keep the cell flow along the bottom surface, and to enhance the contact opportunity between cell-labeled antibodies and protein G. The target antibody binds with the protein G and the number of immobilized yeast cells is counted which reflects the amount of the target antibody. Controlled laminar flow is applied in the device to preferentially remove nonspecifically bound cells, which ensures a high selectivity of the immunoassay. Fig. 2 shows the dose-response curve of the YSD-based microfluidic immunoassay performed on the PDMS-glass chip, with a lower detection limit of 50 ng/mL (S/N = 2).
One of the most significant merits of the YSD-based immunoassay is that the antigen can be synthesized and displayed automatically on the cell surface when the recombinant yeast cells are cultured in galactose containing medium. Thus the tedious steps of antigen purification and wet-chemistry labeling required in a conventional immunoassay are eliminated in the YSD-based  method and the recombinant yeast cells ready for antibody capturing can be collected by simply centrifugation. As a result, easier sample preparation is provided by the YSD-based immunoassay. On the other hand, one of the pivotal issues in the YSD technique is that the functional sites of certain displayed antigens could be inactive due to steric hindrance and alteration in folding, which could potentially limit the application of the YSD-based immunoassay. However, with the YSD technique it is possible to engineer antigenic peptide for improved binding affinity with a specific antibody through display of an antigenic peptide library containing different mutations and quantitative library screening [33,34]. Therefore it is theoretically possible to obtain a functional displayed antigen for any specific antibody. It is anticipated that when combined with antigenic peptide engineering, improved performance of the YSD-based immunoassay could be achieved.

Multiplexed YSD-based microfluidic immunoassay on the PDMS-glass chip
To demonstrate the ability for multiplex detection, different epitopes specific to different target antibodies were displayed on the surface of different yeast cells which simultaneously expressed corresponding intracellular fluorescent proteins. Multiplexed detection was demonstrated using two types of bi-functional yeast cells constructed by our group previously. One of the cells displays a HA tag with an intracellular expression of mCherry ( ex = 587 nm, em = 610 nm) and the other displays a c-myc tag with an intracellular expression of EGFP ( em = 488 nm, em = 507 nm). Hence, the presence and concentration of the anti-HA antibody in the sample are indicated by the number of red cells counted, while the presence and concentration of the anti-c-myc antibody are indicated by the number of green cells counted. Fig. 3 shows the cells captured in the multiplexed detection. The red and green spots represent cells displaying HA tag with an intracellular expression of mCherry and cells displaying c-myc tag with an intracellular expression of EGFP, respectively. When both anti-HA and anti-cmyc were present, a large number of both cells (36 green cells and 37 red cells) were counted. In the absence of both antibodies in the sample, a neglectable number of the cells (7 green cells and 9 red cells) were observed. With a S/N ratio higher than 4, the simultaneous detection of multiple target antibodies was successfully demonstrated with satisfactory selectivity.

YSD-based microfluidic immunoassay on the MPA silicon chip
In order to improve the detection sensitivity of the YSD-based microfluidic immunoassay, a silicon chip incorporating a MPA was used. As shown in Fig. 4, an array of micro pillars with height of 30 m and radius of 15 m was fabricated on a silicon wafer and coated with protein G. Compared to a planar surface, the MPA provides a much higher possibility for the antibody-yeast conjugates to contact with the protein G, which will lead to a higher sensitivity for the detection of the target antibody. An extra advantage brought by the MPA is that the liquid flow can be driven by the capillary force created by the MPA, and therefore the requirement for pumping is eliminated, resulting in an immunoassay that is more compatible with portable applications such as point-of-care testing. The dose-response curve of the YSD-based microfluidic immunoassay performed on the MPA silicon chip is shown in Fig. 5, with a lower detection limit of 5 ng/mL or 1 ng in terms of amount since the sample volume is 200 L (with a signal-to-noise ratio higher than 2), which is 10 times lower than the detection limit on the PDMS-glass chip.
It should be noted that our experimental results indicate that only a very small portion of the injected yeast cells are captured on the chip surface, even when an excessive amount of the target antibody is present (Fig. 5). This probably results from both the low binding efficiency between the target antibody and the yeast surface-displayed antigen, and the low capturing efficiency of target-bound yeast cells by the protein G-modified chip surface. Although the use of a micro pillar array improved the yeast cell capturing efficiency and therefore resulted in lower detection limit, there is still a lot of work to do to further improve the binding and capturing efficiency in order to increase the detection sensitivity.

Conclusion
Utilizing both YSD technique and microfluidic technology, we have developed a prototypical microfluidic immunoassay for sensitive and multiplexed antibody detection. The YSD technique ensures the high sensitivity of the immunoassay, while its implementation on a microfluidic platform eliminates the requirement for the bulky, complex and expensive flow cytometer. Multiplexed YSD-based immunoassay was successfully demonstrated on a PDMS-glass chip, while a MPA-incorporated silicon chip provides a 10 times higher sensitivity and pumping-free operation. This novel YSD-based microfluidic immunoassay is believed to have high potential to be developed into a point-of-care antibody detection tool.