Microarray fabrication techniques for multiplexed bioassay applications.

Microarrays are powerful tools for high-throughput bioassays that can extract information from tens of thousands of micro-spots consisting of biomolecules. This information is invaluable to many applications, such as drug discovery and disease diagnostics. Different applications of these microarrays need spots of different shapes, sizes, and chemistries to achieve their goals. Micro/nano-fabrication techniques are used to make microarrays with different feature structures and array densities for required assay procedures. Understanding these fabrication methods is essential to creating an effective microarray. The purpose of this article is to critically review fabrication methods used in recent microarray-based bioassay studies. We summarized commonly used microarray fabrication techniques and filled the gap in recent literature on relevant topics. We discussed recent examples of how microarrays were fabricated and used in a variety of bioassays. Specifically, we examined microarray printing, various microlithography techniques, and microfluidics-based microarray fabrication. We evaluated how their application shaped the fabrication methods and compared their performance based on different applications. In the end, we discussed current challenges and outlined potential future directions. This review addressed the gap in literature and provided important insights for choosing appropriate fabrication techniques towards different applications.

Compartmentalized Linker Array: A Scalable and Transferrable Microarray Format for Multiplexed Immunoassays.

Microarrays have been widely used for multiplexed bioassays. Fabrication of a conventional microarray typically requires a complex microarray spotter, using which nanoliter bioreagent (e.g., antibody and cells) droplets are delivered onto a glass slide. However, arraying a delicate bioreagent in nanoliter volumes could cause the loss of bioactivity and needs a complex microarray spotter. Further, mixing of different bioreagents in a multiplexed assay leads to cross-reactions, producing false positive signals that impair assay reproducibility and scalability. In this work, we propose a new microarray format, named "compartmentalized linker array (CLA)", that consists of pre-prepared storable microarrays of chemical linkers in microliter compartments. CLA can be used for binding and patterning bioreagents into microarrays by simply pipetting and incubating bioreagent solutions in compartments. Using commonly used aminosilane linker-based antibody microarray, we developed CLA and demonstrated its application for a multiplexed sandwich immunoassay measuring three cancer-related proteins. A "two-phase" blocking system was established for de-activating background regions on glass where no linker molecules are present. Storage conditions of the CLA chip were explored and demonstrated for long-term storage. In a multiplexed immunoassay, low pg/mL sensitivity was achieved for all the three proteins, comparable to those of conventional assays. Moreover, CLA can be potentially used for other applications beyond protein assays, making microarray technology transferrable and widely available for the biological and biomedical research community.

Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids.

Extracellular vesicles (EVs) are nanoscale vesicles secreted from cells, carrying biomolecular cargos similar to their cells of origin. Measuring the protein content of EVs in biofluids can offer a crucial insight into human health and disease. For example, detecting tumor-derived EVs' protein markers can aid in early diagnosis of cancer, which is life-saving. In order to use these EV proteins for diagnosis, sensitive and multiplexed methods are required. The current methods for EV protein detection typically require large sample consumption due to challenges with sensitivity and often need an EV isolation step for complex biofluid samples such as blood plasma. In this work, we have developed a simple and sensitive method for multiplexed detection of protein markers on EV membrane surfaces, which we call "EV dot blotting", inspired by conventional dot blotting techniques. After optimization of multiple factors such as antibody concentration, blocking reagent, type of 3D membranes, and use of gold nanoparticles for signal enhancement, cancer-cell-derived EVs were spiked in pooled normal human plasma for conducting a multiplexed assay in a microarray format. Without the need of isolating EVs from blood plasma, a limit of detection of 3.1 × 105 EVs/mL or 1863 EVs/sample was achieved for CD9 protein, 4.7 × 104 EVs/mL or 281 EVs/sample for CD24, and 9.0 × 104 EVs/mL or 538 EVs/sample for EpCAM, up to 4 orders of magnitude lower than those of conventional ELISA. This platform offers sensitive, multiplexed, simple, and low-cost EV protein detection directly from complex biofluids with minimal sample consumption, providing a useful tool for multiplexed EV protein quantification for a variety of applications.

Measurement of the surface-enhanced coherent anti-Stokes Raman scattering (SECARS) due to the 1574 cm(-1) surface-enhanced Raman scattering (SERS) mode of benzenethiol using low-power (<20 mW) CW diode lasers.

The surface-enhanced coherent anti-Stokes Raman scattering (SECARS) from a self-assembled monolayer (SAM) of benzenethiol on a silver-coated surface-enhanced Raman scattering (SERS) substrate has been measured for the 1574 cm(-1) SERS mode. A value of 9.6 ± 1.7×10(-14) W was determined for the resonant component of the SECARS signal using 17.8 mW of 784.9 nm pump laser power and 7.1 mW of 895.5 nm Stokes laser power; the pump and Stokes lasers were polarized parallel to each other but perpendicular to the grooves of the diffraction grating in the spectrometer. The measured value of resonant component of the SECARS signal is in agreement with the calculated value of 9.3×10(-14) W using the measured value of 8.7 ± 0.5 cm(-1) for the SERS linewidth Γ (full width at half-maximum) and the value of 5.7 ± 1.4×10(-7) for the product of the Raman cross section σSERS and the surface concentration Ns of the benzenethiol SAM. The xxxx component of the resonant part of the third-order nonlinear optical susceptibility |3 χxxxx((3)R)| for the 1574 cm(-1) SERS mode has been determined to be 4.3 ± 1.1×10(-5) cm·g(-1)·s(2). The SERS enhancement factor for the 1574 cm(-1) mode was determined to be 3.6 ± 0.9×10(7) using the value of 1.8×10(15) molecules/cm(2) for Ns.

Measurement of the Raman line widths of neat benzenethiol and a self-assembled monolayer (SAM) of benzenethiol on a silver-coated surface-enhanced Raman scattering (SERS) substrate.

Raman line widths of neat benzenethiol and a self-assembled monolayer (SAM) of benzenethiol on a surface-enhanced Raman scattering (SERS) substrate have been measured using a mini spectrometer with a resolution (full width at half-maximum) of 3.3 ± 0.2 cm(-1). Values of 7.3 ± 0.7, 4.6 ± 0.6, 2.4 ± 0.6, 3.2 ± 0.5, 8.8 ± 0.9, and 11.0 ± 1.1 cm(-1) have been determined for the Raman line widths of the 414, 700, 1001, 1026, 1093, and 1584 cm(-1) modes of neat benzenethiol. Values of 13.3 ± 0.7, 9.1 ± 0.7, 5.1 ± 0.6, 5.9 ± 0.6, 13.3 ± 0.5, and 8.7 ± 0.5 cm(-1) have been determined for the SERS line widths of a benzenethiol SAM on a silver-coated SERS substrate for the corresponding frequency-shifted modes at 420, 691, 1000, 1023, 1072, and 1574 cm(-1). The line widths for the SERS modes at 420, 691, 1000, 1023, and 1072 cm(-1) are about a factor of two larger than those of the corresponding Raman modes. However, the line width of the SERS mode at 1574 cm(-1) is slightly smaller than the corresponding Raman mode at 1584 cm(-1).