Ultrasensitive Detection of PSA Using Antibodies in Crowding Polyelectrolyte Multilayers on a Silicon Nanowire Field-Effect Transistor.

Immunosensors based on field-effect transistors with nanowire channels (NWFETs) provide fast and real-time detection of a variety of biomarkers without the need for additional labels. The key feature of the developed immunosensor is the coating of silicon NWs with multilayers of polyelectrolytes (polyethylenimine (PEI) and polystyrene sulfonate (PSS)). By causing a macromolecular crowding effect, it ensures the "soft fixation" of the antibodies into the 3-D matrix of the oppositely charged layers. We investigated the interaction of prostate-specific antigen (PSA), a biomarker of prostate cancer, and antibodies adsorbed in the PEI and PSS matrix. In order to visualize the formation of immune complexes between polyelectrolyte layers using SEM and AFM techniques, we employed a second clone of antibodies labeled with gold nanoparticles. PSA was able to penetrate the matrix and concentrate close to the surface layer, which is crucial for its detection on the nanowires. Additionally, this provides the optimal orientation of the antibodies' active centers for interacting with the antigen and improves their mobility. NWFETs were fabricated from SOI material using high-resolution e-beam lithography, thin film vacuum deposition, and reactive-ion etching processes. The immunosensor was characterized by a high sensitivity to pH (71 mV/pH) and an ultra-low limit of detection (LOD) of 0.04 fg/mL for PSA. The response of the immunosensor takes less than a minute, and the measurement is carried out in real time. This approach seems promising for further investigation of its applicability for early screening of prostate cancer and POC systems.

Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of ß-Lactamase Genes and Their RNA Transcripts.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to ß-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

Multiplex Digital Quantification of ß-Lactamase Genes in Antibiotic-Resistant Bacteria by Counting Gold Nanoparticle Labels on Silicon Microchips.

Digital quantification based on counting of individual molecules is a promising approach for different biomedical applications due to its enhanced sensitivity. Here, we present a method for the digital detection of nucleic acids (DNA and RNA) on silicon microchips based on the counting of gold nanoparticles (GNPs) in DNA duplexes by scanning electron microscopy (SEM). Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microchips. Then biotin is revealed by a streptavidin-GNP conjugate followed by the detection of GNPs. Sharp images of each nanoparticle allow the visualization of hybridization results on a single-molecule level. The technique was shown to provide highly sensitive quantification of both short oligonucleotide and long double-strand DNA sequences up to 800 bp. The lowest limit of detection of 0.04 pM was determined for short 19-mer oligonucleotide. The method's applicability was demonstrated for the multiplex quantification of several ß-lactamase genes responsible for the development of bacterial resistance against ß-lactam antibiotics. Determination of nucleic acids is effective for both specific DNA in lysates and mRNA in transcripts. The method is also characterized by high selectivity for single-nucleotide polymorphism discrimination. The proposed principle of digital quantification is a perspective for studying the mechanisms of bacterial antibiotic resistance and bacterial response to drugs.

Improvement of Seed-Mediated Growth of Gold Nanoparticle Labels for DNA Membrane-Based Assays.

Gold nanoparticles (AuNPs) are popular labels for colorimetric detection of various analytes, involving proteins, nucleic acids, viruses, and whole cells because of their outstanding optical properties, inertness, and modification variability. In this work, we present an improved approach for enhancement of color intensity for DNA membrane microarrays based on seed-mediated growth of AuNP labels. Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microarrays. Then biotin is revealed by a streptavidin-AuNP conjugate followed by the detection of AuNPs. Optimization of seed-mediated enlargement of AuNPs by the reduction of tetrachloroauric acid with hydroxylamine made it possible to change the coloring of specific spots on the microarrays from pink to a more contrasting black with minor background staining. Mean size of the resulting AuNPs was four times larger than before the enhancement. Adjusting the pH of HAuCl4 solution to 3.5 and use of a large excess of hydroxylamine increased the signal/background ratio by several times. The method's applicability was demonstrated for quantification of a short oligonucleotide of 19 bases and full-length TEM-type ß-lactamase genes of 860 bp responsible for the development of bacterial resistance against ß-lactam antibiotics. Improved protocol for AuNP enlargement may be further transferred to any other membrane-based assays of nucleic acids with both instrumental and visual colorimetric detection.