Evaluating polyvinylidene fluoride - carbon black composites as solid phase microextraction coatings for the detection of urinary volatile organic compounds by gas chromatography-mass spectrometry.

Volatile organic compounds (VOCs) are biomarkers of disease, which can be utilized for accurate diagnostics. The gold standard for VOC identification is gas chromatography-mass spectrometry (GC-MS) as it allows for structure elucidation and quantification. Headspace solid phase microextraction (HS-SPME) is often used in biomarker discovery due to its ability to preconcentrate VOCs prior to GC-MS analysis.

Multiplexed and High-Throughput Label-Free Detection of RNA/Spike Protein/IgG/IgM Biomarkers of SARS-CoV-2 Infection Utilizing Nanoplasmonic Biosensors.

To tackle the COVID-19 outbreak, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an unmet need for highly accurate diagnostic tests at all stages of infection with rapid results and high specificity. Here, we present a label-free nanoplasmonic biosensor-based, multiplex screening test for COVID-19 that can quantitatively detect 10 different biomarkers (6 viral nucleic acid genes, 2 spike protein subunits, and 2 antibodies) with a limit of detection in the aM range, all within one biosensor platform. Our newly developed nanoplasmonic biosensors demonstrate high specificity, which is of the upmost importance to avoid false responses.

Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes.

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels.

Bottom-Up Fabrication of Plasmonic Nanoantenna-Based High-throughput Multiplexing Biosensors for Ultrasensitive Detection of microRNAs Directly from Cancer Patients' Plasma.

There is an unmet need in clinical point-of-care (POC) cancer diagnostics for early state disease detection, which would greatly increase patient survival rates. Currently available analytical techniques for early stage cancer diagnosis do not meet the requirements for POC of a clinical setting. They are unable to provide the high demand of multiplexing, high-throughput, and ultrasensitive detection of biomarkers directly from low volume patient samples ("liquid biopsy").

Enumeration of Rare Cells in Whole Blood by Signal Ion Emission Reactive Release Amplification with Same-Sample RNA Analysis.

Herein is presented a platform capable of detecting less than 30 cells from a whole blood sample by size-exclusion filtration, microfluidic sample handling, and mass spectrometric detection through signal ion emission reactive release amplification (SIERRA). This represents an approximate 10-fold improvement in detection limits from previous work. Detection by SIERRA is accomplished through the use of novel nanoparticle reagents coupled with custom fluidic fixtures for precise sample transfer.

Heat-enhanced peptide synthesis on Teflon-patterned paper.

In this report, we describe the methodology for 96 parallel organic syntheses of peptides on Teflon-patterned paper assisted by heating with an infra-red lamp. SPOT synthesis is an important technology for production of peptide arrays on a paper-based support for rapid identification of peptide ligands, epitope mapping, and identification of bio-conjugation reactions. The major drawback of the SPOT synthesis methodology published to-date is suboptimal reaction conversion due to mass transport limitations in the unmixed reaction spot.

Parallel Syntheses of Peptides on Teflon-Patterned Paper Arrays (SyntArrays).

Screening of peptides to find the ligands that bind to specific targets is an important step in drug discovery. These high-throughput screens require large number of structural variants of peptides to be synthesized and tested. This chapter describes the generation of arrays of peptides on Teflon-patterned sheets of paper.

Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences.

Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout.

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-β-D-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo(®)) β-galactosidase substrates were tested in the assay.

Flow-through synthesis on Teflon-patterned paper to produce peptide arrays for cell-based assays.

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis.

Antimicrobial susceptibility assays in paper-based portable culture devices.

To detect antibiotic-resistant bacteria in areas remote from microbiology laboratories, we designed portable culture devices performing an analogue of the Kirby-Bauer disk diffusion test inside patterned papers embedded in tape. We quantified the antibiotic susceptibility of several strains of Escherichia coli and Salmonella typhimurium by measuring blue-colored zones of inhibited growth.

Platform for high-throughput testing of the effect of soluble compounds on 3D cell cultures.

In vitro 3D culture could provide an important model of tissues in vivo, but assessing the effects of chemical compounds on cells in specific regions of 3D culture requires physical isolation of cells and thus currently relies mostly on delicate and low-throughput methods. This paper describes a technique ("cells-in-gels-in-paper", CiGiP) that permits rapid assembly of arrays of 3D cell cultures and convenient isolation of cells from specific regions of these cultures. The 3D cultures were generated by stacking sheets of 200-μm-thick paper, each sheet supporting 96 individual "spots" (thin circular slabs) of hydrogels containing cells, separated by hydrophobic material (wax, PDMS) impermeable to aqueous solutions, and hydrophilic and most hydrophobic solutes.

Functionalization of optical nanotip arrays with an electrochemical microcantilever for multiplexed DNA detection.

Optical nanotip arrays fabricated on etched fiber bundles were functionalized with DNA spots. Such unconventional substrates (3D and non-planar) are difficult to pattern with standard microfabrication techniques but, using an electrochemical cantilever, up to 400 spots were electrodeposited on the nanostructured optical surface in 5 min. This approach allows each spot to be addressed individually and multiplexed fluorescence detection is demonstrated.

Opto-electrochemical nanosensor array for remote DNA detection.

A high-density array of opto-electrochemical nanosensors is presented for remote DNA detection. It was fabricated by chemical etching of a coherent optical fibre bundle to produce a nanotip array. The surface of the etched bundle was sputter-coated with a thin ITO layer which was eventually insulated by an electrophoretic paint.

Integration of paper-based microfluidic devices with commercial electrochemical readers.

The combination of simple Electrochemical Micro-Paper-based Analytical Devices (EµPADs) with commercially available glucometers allows rapid, quantitative electrochemical analysis of a number of compounds relevant to human health (e.g., glucose, cholesterol, lactate, and alcohol) in blood or urine.

Lithography by Scanning Electrochemical Microscopy with a multiscaled electrode.

A multiscaled electrochemical probe is presented for Scanning Electrochemical Microscopy (SECM) experiments. It is fabricated by wet chemical etching followed by sputter-coating of an ordered optical fiber bundle. Owing to the optical fiber bundle preparation, the global electrode may present different shapes.

Optical-fiber-microsphere for remote fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a versatile method that would greatly benefit to remote optical-fiber fluorescence sensors. However, the current state-of-the-art struggles with high background and low detection sensitivities that prevent the extension of fiber-based FCS down to the single-molecule level. Here we report the use of an optical fiber combined with a latex microsphere to perform FCS analysis.

Multiplexed sandwich immunoassays using electrochemiluminescence imaging resolved at the single bead level.

A new class of bead-based microarray that uses electrogenerated chemiluminescence (ECL) as a readout mechanism to detect multiple antigens simultaneously is presented. This platform demonstrates the possibility of performing highly multiplexed assays using ECL because all the individual sensing beads in the array are simultaneously imaged and individually resolved by ECL. Duplex and triplex assay results are demonstrated as well as a cross reactivity study.