Three-dimensional protein microarrays fabricated on reactive microsphere modified COC substrates.

Fabrication of three-dimensional (3D) surface structures for the high density immobilization of biomolecules is an effective way to prepare highly sensitive biochips. In this work, a strategy to attach polymeric microspheres on a cyclic olefin copolymer (COC) substrate for the preparation of a 3D protein chip was developed. The COC surface was firstly functionalized by the photograft technique with epoxy groups, which were subsequently converted to amine groups. Then monodisperse poly(styrene-alt-maleic anhydride) (PSM) copolymer microspheres were prepared by self-stabilized precipitation polymerization and deposited as a single layer on a modified COC surface to form a 3D surface texture. The surface roughness of the COC support undergoes a significant increase from 1.4 nm to 37.1 nm after deposition of PSM microspheres with a size of 460 nm, and the modified COC still maintains a transmittance of more than 63% at the fluorescence excitation wavelengths (555 nm and 647 nm). The immobilization efficiency of immunoglobulin G (IgG) on the 3D surface reached 75.6% and the immobilization density was calculated to be 0.255 µg cm-2, at a probe protein concentration of 200 µg mL-1. The 3D protein microarray can be rapidly blocked by gaseous ethylenediamine within 10 minutes due to the high reactivity of anhydride groups in PSM microspheres. Immunoassay results show that the 3D protein microarray achieved specific identification of the target protein with a linear detection range from 6.25 ng mL-1 to 250 ng mL-1 (R2 > 0.99) and a limit of detection of 8.87 ng mL-1. This strategy offers a novel way to develop high performance polymer-based 3D protein chips.

Systematic and Rational Design of Protein Arrays in Noncontact Printers: Pipeline and Critical Aspects.

The systematic design and construction of customized protein microarrays are critical for the further successful screening of biological samples in biomedical research projects. In general protein microarrays are classified according to the content, detection method, and printing methodology, among others. Here, we are focused on the type of printing: contact and noncontact. Both approaches have advantages and disadvantages; however, in any of the approaches, a prior well design and systematic preparation of materials and/or instruments required for the customized antibody arrays is critical. In this chapter, the process for an antibody microarray by a noncontact printer is described in detail from the preparation of array content to the analysis, including quality control steps.

Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip.

The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein-DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.

Anaerobic Stopped-Flow Spectrophotometry with Photodiode Array Detection in the Presteady State: An Application to Elucidate Oxidoreduction Mechanisms in Flavoproteins.

Anaerobic stopped-flow (SF) spectrophotometry is a powerful biophysical tool that allows a complete kinetic characterization of protein interactions with other molecules when they are in different redox states, as well as of the redox processes consequence of such interactions. Differences in the absorption spectroscopic properties of oxidized, semiquinone and hydroquinone states of flavoproteins, as well as the appearance of transient spectroscopic features produced by the flavin cofactor during substrate binding and electron transfer processes, have made SF a suitable technique for kinetically dissecting their mechanisms of reaction. In addition, SF coupled to photodiode array detection, enables kinetic data collection in a wavelength range. When such type of data are available for a flavoprotein reaction, they allow for obtaining detailed information of individual reaction steps, including intermolecular dissociation constants as well as electron transfer rate constants. Methodologies for the mechanistic characterization of flavoproteins involved in redox processes by SF spectrophotometry are described in this chapter.

Highly Sensitive Simultaneous Detection of Multiple Mycotoxins Using a Protein Microarray on a TiO2-Modified Porous Silicon Surface.

A new protein microarray method for multiplex mycotoxin detection in parallel has been established on a stable TiO2-modified porous silicon (PSi) surface. A typical competitive immunoassay microarray protocol has been developed for simultaneous detection of multiplex mycotoxins including aflatoxin B1 (AFB1), ochratoxin A (OTA), and fumonisin B1 (FB1) on the TiO2-PSi surface. The epoxy groups were selected to modify the surface of a TiO2-PSi wafer for the immobilization of artificial antigens of mycotoxins because of their high signal-to-noise ratios. Under optimal conditions, the developed method showed wide linear detection ranges of 0.01-1 ng/mL for OTA, 0.001-1 ng/mL for AFB1, and 0.01-1 ng/mL for FB1 and low limit of detections (LODs) of 0.433 ng/mL for OTA, 0.243 ng/mL for AFB1, and 0.093 ng/mL for FB1. The microarray method can specifically identify the three mycotoxins and their analogues. The recovery rates in real samples were within 75-120%, which were in agreement with that of the classical ELISA method. The new method has great application potential for rapid, sensitive, and high-throughput screening of multiplex mycotoxins and other target molecules.

Surface Plasmon Resonance Imaging-Mass Spectrometry Coupling on Antibody Array Biochip: Multiplex Monitoring of Biomolecular Interactions and On-Chip Identification of Captured Antigen.

The coupling of surface plasmon resonance imaging (SPRi) with mass spectrometry (MS) offers a very promising multidimensional analysis. This system takes advantage of the two well-established techniques: SPR, which allows for the analysis of biomolecular interactions through the determination of kinetic and thermodynamic constants, and MS, which can characterize biological structures from mass measurements and fragmentation experiments. Here, a protocol for the coupling of SPRi with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described using a biochip grafted by antibodies in an array format. Interaction between ß-lactoglobulin antibodies and the protein antigen is detected and analyzed by SPRi. Then, the arrayed biochip which fitted a commercially MALDI target was inserted in a MALDI source, and mass spectra were recorded directly from the biochip surface from each antibody spot, showing protein ions attributed to the corresponding specific protein antigens.

Multiplexed Protein Biomarker Detection with Microfluidic Electrochemical Immunoarrays.

Electrochemistry is a multidisciplinary field encompassing the study of analytes in solution for detection and quantification. For the medical field, this brings opportunities to the clinical practice of disease detection through measurements of disease biomarkers. Specifically, panels of biomarkers offer an important future option that can enable physicians' access to blood, saliva, or urine bioassays for screening diseases, as well as monitoring the progression and response to therapy. Here, we describe the simultaneous detection of eight protein cancer biomarkers in a 30-min assay by a microfluidic electrochemical immunoarray.

Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.

Rapid Fabrication of Protein Microarrays via Autogeneration and on-Chip Purification of Biotinylated Probes.

A streamlined approach toward the rapid fabrication of streptavidin-biotin-based protein microarrays was investigated. First, using our engineered versatile plasmid (pBADcM-tBirA) and an optimal coexpression strategy for biotin ligase and biotin acceptor peptide (BAP) chimeric recombinant protein, an autogeneration system for biotinylated probes was developed. This system permitted an advantageous biotinylation of BAP chimeric recombinant proteins, providing a strategy for the high-throughput synthesis of biotinylated probes. Then, to bypass the conventional rate-limiting steps, we employed an on-chip purification process to immobilize the biotinylated probes with high-throughput recombinant lysates. The integration of the autogeneration of probes and on-chip purification not only contributed to the effective and reliable fabrication of the protein microarray, but also enabled simplification of the process and an automated throughput format. This labor- and cost-effective approach may facilitate the use of protein microarrays for diagnosis, pharmacology, proteomics, and other laboratory initiatives.

Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.

Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of a Systemic Inflammatory Response Syndrome (SIRS) in human serum.

The Systemic Inflammatory Response Syndrome (SIRS), a sepsis related inflammatory state, is a self-defense mechanism against specific and nonspecific stimuli. The six most extensively studied inflammatory biomarkers for the clinical diagnosis of SIRS are interleukin 4 (hIL-4), interleukin 6 (hIL-6), interleukin 10 (hIL-10), tumor necrosis factor alpha (hTNF-α), interferon gamma (hIFN-γ) and procalcitonin (hPCT). These biomarkers are naturally present (but usually only at low concentration) in SIRS infected patients [1, 2] and thus the development of a highly sensitive detection method is of major clinical interest. However, the existing analytical techniques are lacking in required analytical sensitivity and parallel determination of these biomarkers. We developed a fast, easy and cost-efficient protein microarray biochip where the capture molecules are attached on hydrogel spots, enabling SIRS diagnosis by parallel detection of these six clinically relevant biomarkers with a sample volume of 25 µl. With our hydrogel based protein microarray biochip we achieved a limit of detection for hIL-4 of 75.2 pg/ml, for hIL-6 of 45.1 pg/ml, for hIL-10 of 71.5 pg/ml, for hTNF-α of 56.7 pg/ml, for IFN-γ of 46.4 pg/ml and for hPCT of 1.1 ng/ml in spiked human serum demonstrating sufficient sensitivity for clinical usage. Additionally, we demonstrated successful detection of two relevant SIRS biomarkers in clinical patient samples with a turnaround time of the complete analysis from sample-to-answer in less than 200 minutes.

High Precision RPPA: Concept, Features, and Application Performance of the Integrated Zeptosens Platform.

An integrated reverse phase protein array (RPPA) platform shall allow the precise monitoring of expression level and changes of proteins and their functional states in a highly parallel manner even when samples exhibit a complex matrix like in tumor tissues and are available only in very limited amounts. Ideally the full workflow from sample preparation to data visualization shall be covered.This book chapter describes the key elements of the integrated Zeptosens RPPA platform. It addresses critical platform and process design requirements, considerations, and elements as well as critical process steps and quality aspects. Sophisticated instrumentation, high sensitivity readout, and dedicated chip and assay handling equipment act in concert with streamlined protocols, optimal reagents, and dedicated lab equipment in the hands of trained users to achieve an outstanding overall performance of the realized system. Based on results from comprehensive signaling protein and pathway profiling studies targeted for preclinical drug efficacy testing and development, it gives an overview of application performance by means of coefficients of variation (CVs) that can be achieved for assay signals from technical and biological sample replicates with this state-of-the-art integrated RPPA platform and process.The Zeptosens RPPA platform has proven to provide valuable biological information with a high level of confidence and has shown its validity in generating sound mechanistic as well as prognostic and predictive information when analyzing cell and tissue materials on the functional protein level.

Solid Pin Protein Array Printing Platforms.

Reverse phase protein arrays (RPPA) are miniature dot blots constructed using robotic arrayers to deposit protein containing samples onto nitrocellulose-coated glass slides. Reverse phase protein arrays address the challenge of quantifying low-abundance proteins and posttranslationally modified proteins in cellular lysates and body fluids. RPPA technology is ideally suited to biomarker discovery, signal pathway profiling, functional phenotype analysis, and mechanism of action studies for drug discovery. Each array is fabricated with specimens, controls, and calibrators, thus providing a complete assay on each slide. Constructing a reverse phase protein array initially consists of selecting an arrayer, pin type, print head configuration, and nitrocellulose slide that is optimized for the particular specimen type and protein detection method. Herein we present the nuances of RPPA fabrication and study design using a solid pin arrayer and nitrocellulose-coated slides.

Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-Dimensional Peptide Arrays to Multiplex Bead Flow Assays.

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

Disease antigens detection by silicon nanowires with the efficiency optimization of their antibodies on a chip.

Enhancing the efficiency of antibody protein immobilized on a silicon nanowire-based chip for their antigens detection is reported. An external electric field (EEF) is applied to direct the orientation of antibodies during their immobilization on a chip. Atomic force microscopy (AFM) is used to measure the binding forces between immobilized antibody and targeting antigen under the influence of EEF at different angles. The maximum binding force under a specific angle (optimal angle; oa) of EEF (maxEEFoa) implies the optimal orientation of the antibodies on the chip. In this report, two different cancer carcinoembryonic antigen (CEA)-related cell adhesion molecules 5 (CEACAM5) & 1 (CEACAM1) were used for the examples of disease antigen detection. maxEEFoa of anti-CEACAM5 or anti-CEACAM1 immobilized on a general chip was firstly determined. Spectroscopy of AFM revealed that both binding forces were the largest ones with their antigens when maxEEFoa was applied as compared with no or other angles of EEF. These antibody proteins accompanied with the application of EEF were secondly immobilized on silicon-nanowires (n = 1000) and the field effects were measured (∆I) as their target antigens were approached. Results showed that ∆I was the largest ones when maxEEFoas (225°/270° and 135°/180° for anti-CEACAM5 and anti-CEACAM1, respectively) were applied as compared with other angles of EEF. These observations imply that the silicon nanowires together with the application of maxEEFoa as detection tools could be applied for the cancer diagnostics in the future.

Improved methods for the detection of histone interactions with peptide microarrays.

Histone post-translational modifications contribute to chromatin function largely through the recruitment of effector proteins that contain specialized "reader" domains. While a significant number of reader domains have been characterized for their histone binding specificities, many of these domains remain poorly characterized. Peptide microarrays have been widely employed for the characterization of histone readers, as well as modifying enzymes and histone antibodies. While powerful, this platform has limitations in terms of its sensitivity and they frequently miss low affinity reader domain interactions. Here, we provide several technical changes that improve reader domain detection of low-affinity interactions. We show that 1% non-fat milk in 1X PBST as the blocking reagent during incubation improved reader-domain interaction results. Further, coupling this with post-binding high-salt washes and a brief, low-percentage formaldehyde cross-linking step prior to the high-salt washes provided the optimal balance between resolving specific low-affinity interactions and minimizing background or spurious signals. We expect this improved methodology will lead to the elucidation of previously unreported reader-histone interactions that will be important for chromatin function.

Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip.

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.

Graphically encoded suspension array for multiplex immunoassay and quantification of autoimmune biomarkers in patient sera.

In precision medicine, clinical decisions and pharmaceutical evaluations tends to be made upon parallel analysis of multiple protein biomarkers. Currently, the growing needs of high-throughput multiplex immunoassay is partially satisfied by spectrally encoded bead flow suspension arrays and other platforms, yet there is still room for progress in terms of encoding capacity, decoding accuracy, ease-of-manufacture/operation, and cost-effectiveness, for which graphical suspension arrays could make substantial contributions. Here we described a suspension array system made up of graphically encoded silica particles, an automated microplate imager and an in-house data processing program. The micro-fabricated, highly uniform planar particles provide a code space of 128-plex with further extendibility. The derived multiplex immunoassay reaches sub-picogram per milliliter sensitivity level (lowest LoD = 80 fg/ml) with wide dynamic range, as well as high precision and accuracy. The potential of clinical diagnostics was demonstrated by parallel measurement of three serum biomarkers for type 1 diabetes patients. Importantly, use of standard microplates as assay vessel extends its power to high-throughput applications, such as disease screening or drug discovery.

A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.

We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.

Label-free Bacteria Quantification in Blood Plasma by a Bioprinted Microarray Based Interferometric Point-of-Care Device.

Existing clinical methods for bacteria detection lack speed, sensitivity, and, importantly, point-of-care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Here we report a portable PoC device based on lens-free interferometric microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria ( E. coli) quantification. With only one-step sample handling we offer a sample-to-data turnaround time of 40 min. Our technology features detection sensitivity of a single bacterial cell both in buffer and in diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and noninfectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.