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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 ( R 2 > 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.

A new protein microarray method for multiplex mycotoxin detection in parallel has been established on a stable TiO 2 -modified porous silicon (PSi) surface. A typical competitive immunoassay microarray protocol has been developed for simultaneous detection of multiplex mycotoxins including aflatoxin B 1 (AFB 1 ), ochratoxin A (OTA), and fumonisin B 1 (FB 1 ) on the TiO 2 -PSi surface. The epoxy groups were selected to modify the surface of a TiO 2 -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 AFB 1 , and 0.01-1 ng/mL for FB 1 and low limit of detections (LODs) of 0.433 ng/mL for OTA, 0.243 ng/mL for AFB 1 , and 0.093 ng/mL for FB 1 . 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.

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.

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 β-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.

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.

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.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 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.

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., Competing Interests: The authors have declared that no competing interests exist.

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 ( max EEF oa ) 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. max EEF oa 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 max EEF oa 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 max EEF oa s (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 max EEF oa as detection tools could be applied for the cancer diagnostics in the future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 × 10 4 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 × 10 5 molecular variants of full-length human O 6 -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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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.

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.

Alzheimer's disease (AD) is a common neurodegenerative disorder in elderly people, and is associated with a heavy financial burden on our society. The use of serologic biomarkers is an attractive method to diagnose AD. Although the determination of blood-based biomarkers for AD has been explored in many studies, few practical diagnosis methods have been used in the clinic. In this work, we constructed a "chemical tongue" sensor array that is easy to use and based on four kinds of fluorescent gold nanoclusters (Au NCs) for discriminating between multiple proteins at nanomolar concentrations. The device utilizes a linear discrimination analysis based on fluorescence intensity response patterns. Using this chemical tongue sensor array, multiple proteins can be confidently identified even in complex biological systems, such as human urine. Most importantly, sera of AD patients could be effectively discriminated from those of osteoarthritis patients, or of healthy people. Also, the results obtained for the AD patients by the chemical tongue sensor array were validated by CSF determination. We conclude that the chemical tongue sensor array manufactured in this work paves the way for designing an auxiliary diagnosis method for AD that is less invasive and more convenient for the large-scale screening of patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Ideal monitoring devices should enjoy a combination of characteristics, e.g. high sensitivity, multiplexing, portability, short time-to-result (TTR). Typically, no device meets all of these demands since some of them are contradictory, to some extent. Herein, we present a miniaturized platform based on fluorescent detection, which is sensitive, readily allows multiplexing, and allows real-time monitoring of the signal, thus allowing extraction of kinetic information as well as drastic reduction of TTR. This is achieved via miniaturization of active spots, integration with microfluidics, and algorithmic approaches. We validate its performance by comparing with evanescent field excitation, which obtains similar results, however without the addition of the necessary complex hardware., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The detection of body fluids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identification system for body fluids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced fluorescence (MEF) substrate for semen and vaginal fluid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the fluorescence signal was saturated. To assess the performance of the developed identification chip, the identification of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identification (RSID) Semen kit., Competing Interests: The authors declare no conflicts of interest.

Antibody reagents have been remained as a standard approach to characterize blood group (BG) antigens in clinic. The specificity and cross-reactivity of these BG antibodies are routine detected using the gel microcolumn assay (GMA). However, the GMA is neither specific nor sensitive, thus increasing the risk of improperly matched RBC transfusions. In this work, we describe a bead-based RBC membrane antigen array to detect BG antibody-antigen binding with ∼700-fold higher sensitivity and dynamic range than the GMA. RBC membrane antigen arrays were fabricated using fragmented RBC membranes highly enriched in BG panel antigens. The arrays were then used to screen the interactions of 15 BG reagents to three antigen panels. The majority of the antibody reactions (i.e., 86.7%; 39/45) aligned with those obtained with the GMA. The six cross-reactive, nonspecific antibody reactions identified only by our arrays (i.e., 13.3%; 6/45) were confirmed by agglutination inhibition and genotyping assays. These results demonstrate that our RBC membrane antigen array has great potential in screening BG antibodies and improving the safety of RBC transfusions.

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in "universal" flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

Multiplex assays for autoantibodies have shown utility both in research towards understanding the basic biology of autoimmune disease, and as tools for clinical diagnosis. New label-free multiplex analysis methods have the potential to streamline both the process of assay development and assay workflow. We report fabrication and testing of a 5-plex autoantigen microarray using the Arrayed Imaging Reflectometry (AIR) platform. This label-free technology provides rapid, sensitive, and quantitative detection of an arbitrary number of analytes in a standard multiwell format. In this work, we demonstrate that AIR is able to detect antibodies to Ro60, La/SSB, Scl-70, BicD2, and Ro52 in single-donor human serum samples with multiplex results comparable to singleplex ELISA or Luminex assays., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm 2 . To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rhinovirus (RV) infections are major triggers of acute exacerbations of severe respiratory diseases such as pre-school wheeze, asthma and chronic obstructive pulmonary disease (COPD). The occurrence of numerous RV types is a major challenge for the identification of the culprit virus types and for the improvement of virus type-specific treatment strategies. Here, we develop a chip containing 130 different micro-arrayed RV proteins and peptides and demonstrate in a cohort of 120 pre-school children, most of whom had been hospitalized due to acute wheeze, that it is possible to determine the culprit RV species with a minute blood sample by serology. Importantly, we identify RV-A and RV-C species as giving rise to most severe respiratory symptoms. Thus, we have generated a chip for the serological identification of RV-induced respiratory illness which should be useful for the rational development of preventive and therapeutic strategies targeting the most important RV types.

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

Glass slides coated with a poly(methyl methacrylate) layer and plasma micro-nanotextured to acquire 3D topography (referred as 3D micro-nanotextured slides) were evaluated as substrates for biomolecule microarrays. Their performance is compared with that of epoxy-coated glass slides. We found that the proposed three-dimensional (3D) slides offered significant improvements in terms of spot intensity, homogeneity, and reproducibility. In particular, they provided higher spot intensity, by a factor of at least 1.5, and significantly improved spot homogeneity when compared to the epoxy-silane coated ones (intra-spot and between spot coefficients of variation ranging between 5 and 15% for the 3D micro-nanotextured slides and between 25 and 85% for the epoxy-silane coated ones). The latter was to a great extent the result of a strong "coffee-ring" effect observed for the spots created on the epoxy-coated slides; a phenomenon that was severely reduced in the 3D micro-nanotextured slides. The 3D micro-nanotextured slides offered in addition higher signal to noise ratio values over a wide range of protein probe concentrations and shelf-life over one year without requirement for specific storage conditions. Finally, the protocols employed for protein probe immobilization were extremely simple., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nonspecific binding or adsorption of biomolecules presents as a major obstacle to higher sensitivity, specificity and reproducibility in microarray technology. We report herein a method to fabricate antifouling microarray via photopolymerization of biomimetic betaine compounds. In brief, carboxybetaine methacrylate was polymerized as arrays for protein sensing, while sulfobetaine methacrylate was polymerized as background. With the abundant carboxyl groups on array surfaces and zwitterionic polymers on the entire surfaces, this microarray allows biomolecular immobilization and recognition with low nonspecific interactions due to its antifouling property. Therefore, low concentration of target molecules can be captured and detected by this microarray. It was proved that a concentration of 10ngmL -1 bovine serum albumin in the sample matrix of bovine serum can be detected by the microarray derivatized with anti-bovine serum albumin. Moreover, with proper hydrophilic-hydrophobic designs, this approach can be applied to fabricate surface-tension droplet arrays, which allows surface-directed cell adhesion and growth. These light controllable approaches constitute a clear improvement in the design of antifouling interfaces, which may lead to greater flexibility in the development of interfacial architectures and wider application in blood contact microdevices., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 10 6 CFU/ml) and Francisella tularensis (LOD of 1 × 10 4 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents., (Copyright © 2018 American Society for Microbiology.)

This report describes a novel method for isolating and detecting individual enzyme molecules using polymer arrays of picoliter microwells. A fluidic flow-cell device containing an array of microwells is fabricated in cyclic olefin polymer (COP). The use of COP microwell arrays simplifies experiments by eliminating extensive device preparation and surface functionalization that are common in other microwell array formats. Using a simple and robust loading method to introduce the reaction solution, individual enzyme molecules are trapped in picoliter microwells and subsequently isolated and sealed by fluorinated oil. The sealing is stable for hours in the COP device. The picoliter microwell device can measure enzyme concentrations in the low-femtomolar range by counting the number of active wells using a digital read-out. These picoliter microwell arrays can also easily be regenerated and reused.

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.0) and an advanced metabolizing enzyme microarray (MetaChip 2.0). The DataChip is a functionalized micropillar chip that supports the Hep3B human hepatoma cell line in a 3D microarray format. The MetaChip is a microwell chip containing immobilized DMEs found in the human liver. As a proof of concept for generating compound metabolites in situ on the chip and rapidly assessing their toxicity, 22 model compounds were dispensed into the MetaChip and sandwiched with the DataChip. The IC 50 values obtained from the chip platform were correlated with rat LD 50 values, human C max values, and drug-induced liver injury categories to predict adverse drug reactions in vivo. As a result, the platform had 100% sensitivity, 86% specificity, and 93% overall predictivity at optimum cutoffs of IC 50 and C max values. Therefore, the DataChip/MetaChip platform could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.

Quantification of single-cell proteomics provides key insights in the field of cellular heterogeneity. This chapter discusses the emerging techniques that are being used to measure the protein copy numbers at the single-cell level, which includes flow cytometry, mass cytometry, droplet cytometry, microengraving, and single-cell barcoding microchip. The advantages and limitations of each technique are compared, and future research opportunities are highlighted.

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, the CA125 protein purified from ovarian cancer cell lines, and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glycobiomarker development., (© 2018 Elsevier Inc. All rights reserved.)

The technique of surface-enhanced laser desorption/ionization time-of-flight SELDI-TOF mass spectrometry could be used to establish serum or plasma proteomic profiles in esophageal adenocarcinoma. The protein profiles in patients may be used to predict survival and monitor response to chemotherapy in patients with esophageal adenocarcinoma. Here, a protocol for sample preparation from esophageal adenocarcinoma, analysis of proteomic profiles, and collection of protein fractions for identification of significant peaks is presented.

Because of our access to human genome data and ever-improving genome sequencing and proteome analysis methods, we are much better in terms of our understanding of biological processes. In addition to genomics, proteomics, and other "omics" methods, availability of more sophisticated molecular assaying methods has augmented our knowledge about immune processes toward auto- and allogeneic targets. High-density protein arrays are developed to analyze protein-small molecule interactions, enzyme-substrate profiling, protein-protein interaction, and immune monitoring by assessing antibodies in the serum.

Microarrays are powerful experimental tools for high-throughput screening of cellular behavior in multivariate microenvironments. Here, we present a new, facile and rapid screening method for probing cellular behavior in 3D tissue microenvironments. This method utilizes a photoclickable peptide microarray platform developed using electrospun fibrous poly(ethylene glycol) hydrogels and microarray contact printing. We investigated the utility of this platform with five different peptide motifs and ten cell types including stem, terminally differentiated, cancer or immune cells that were from either primary origin or cell lines and from different species. We validated the capabilities of this platform to screen arrays consisting of multiple peptide motifs and concentrations for selectivity to cellular adhesion and morphology. Moreover, this platform is amenable to controlled spatial presentation of peptides. We show that by leveraging the differential attachment affinities for two cell types to two different peptides, this platform can also be used to investigate cell-cell interactions through miniature co-culture peptide arrays. Our fibrous peptide microarray platform enables high-throughput screening of 3D tissue microenvironments in a facile and rapid manner to investigate cell-matrix interactions and cell-cell signaling and to identify optimal tissue microenvironments for cell-based therapies., (Copyright © 2017. Published by Elsevier Ltd.)

Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication.

Background: Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG 4 , as a potential parameter for tolerance development, remain to be optimized., Objective: We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG 4 , and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG 4 were assessed., Methods: Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG 4 . A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG 4 -specific fluorescence on silicon microarrays., Results: Allergen-specific IgE and IgG 4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kU A /L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG 4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results., Conclusions: We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research., (© 2017 John Wiley & Sons Ltd.)

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Characterization of RNA-binding protein interactions with RNA became inevitable to properly understand the cellular mechanisms involved in gene expression regulation. Structural investigations bring information at the atomic level on these interactions and complementary methods such as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are commonly used to quantify the affinity of these RNA-protein complexes and evaluate the effect of mutations affecting these interactions. The switchSENSE technology has recently been developed and already successfully used to investigate protein interactions with different types of binding partners (DNA, protein/peptide or even small molecules). In this study, we show that this method is also well suited to study RNA binding proteins (RBPs). We could successfully investigate the binding to RNA of three different RBPs (Fox-1, SRSF1 and Tra2-β1) and obtained K D values very close to the ones determined previously by SPR or ITC for these complexes. These results show that the switchSENSE technology can be used as an alternative method to study protein-RNA interactions with K D values in the low micromolar (10 -6 ) to nanomolar (10 -7 -10 -9 ) and probably picomolar (10 -10 -10 -12 ) range. The absence of labelling requirement for the analyte molecules and the use of very low amounts of protein and RNA molecules make the switchSENSE approach very attractive compared to other methods. Finally, we discuss about the potential of this approach in obtaining more sophisticated information such as structural conformational changes upon RBP binding to RNA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Hemorrhage is the most important complication of antithrombotic therapy with P2Y12 receptor blockers. The administration of platelet concentrates (PCs) and von Willebrand factor (vWF) concentrates are common procedures to normalize impaired primary hemostasis in bleeding patients. We tested whether this strategy reverses the effect of clopidogrel using a parallel plate flow chamber model., Methods: Whole blood from patients, who received a loading dose of clopidogrel with 600 mg and an ongoing dual antiplatelet therapy with 75 mg/d clopidogrel and 100 mg/d acetyl salicylic acid, compared with blood from healthy volunteers was examined in a collagen-coated parallel plate flow chamber. Blood was perfused by suction at a shear rate of 300/s, which is equivalent to 14 dynes/cm to resemble shear stress in conduit arteries. Platelet-covered area, individual thrombus size, and the average thrombus size were assessed morphometrically. The equivalent of 2 or 5 units of PC and/or 2 U/mL of vWF concentrate were used in an attempt to restore coagulation capacity in blood samples of clopidogrel-treated patients., Results: In this model, clopidogrel reduced the increase of thrombus size. The equivalent of 2 U of PC or 2 U/mL of vWF alone did not show any significant changes in thrombus size. 5 U of PC increased thrombus size in clopidogrel-treated patients (P < .05). Thrombus size in clopidogrel blood was increased by combined PC and vWF treatment (by 50%, P < .05), but this increase did not reach control levels (P < .05)., Conclusions: This flow chamber model is suitable for detection of the antiplatelet effect of clopidogrel. Ex vivo addition of PC or vWF does not overcome the effects of clopidogrel in this model, but the combination of both shows a mild and significant improvement in thrombus size.

Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10×10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects., (Copyright © 2016. Published by Elsevier Inc.)

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Peptides homing tumor vasculature are considered promising molecular imaging agents for cancer detection at an early stage. In addition to their high binding affinity, improved tissue penetrating ability, and low immunogenicity, they can deliver targeted anticancer drugs, thus expanding therapeutic treatments. Among those, CREKA, a linear peptide that specifically binds to clotted-plasma proteins in tumor vessels, has been recently employed to design bioactive systems able to target different cancer types. Within this context, this paper explores the biorecognition event between CR(NMe)EKA, an engineered CREKA-analog bearing a noncoded amino acid (N-methyl-Glu) that is responsible for its enhanced activity, and clotted-plasma proteins (fibrin and fibrinogen) by nanomechanical detection. Specifically, the tumor-homing peptide was covalently attached via epoxysilane chemistry onto silicon microcantilever chips that acted as sensors during dynamic mode experiments. Before that, each step of the functionalization process was followed by contact angle measurements, interferometry, X-ray photoelectron spectroscopy, and atomic force microscopy, thus revealing the applied protocol as a suitable strategy. The fibrin(ogen)-binding induced by CR(NMe)EKA was detected by the resonance frequency shift of the cantilevers, and a detection limit of 100 ng/mL was achieved for both proteins. Even though further development is required, this work reflects the promising application of emerging technologies capable of assisting in the comprehension of biological interactions and their implications in the biotechnological field. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)