Elena Nikolaevna Savvateeva

Gel-based microarrays in clinical diagnostics in Russia

Immobilization of molecular probes in 3D hydrogel elements provides some essential advantages compared with conventional flat surfaces. In this article, an integrated technology based on the use of low-density microarrays comprised of hemispherical gel elements, developed at the Engelhardt Institute of Molecular Biology (Moscow, Russia) for various applications will be reviewed. The structure of the gel can be adapted for immobilization of virtually any biological molecules in a natural hydrophilic environment. The discrimination between matching and mismatching duplexes of nucleic acids in these conditions is more reliable than on conventional flat surfaces, minimizing the number of elements needed to detect specific sequences. Protein molecules immobilized in hydrogel-based biochips better preserve their biological properties. As described in this article, such biochips were successfully applied for laboratory diagnostics in a wide variety of clinical conditions involving the identification of bacterial and viral pathogens, cancer-related mutations and protein tumor markers.

Hydrogel-based protein and oligonucleotide microchips on metal-coated surfaces: enhancement of fluorescence and optimization of immunoassay.

Manufacturing of hydrogel-based microchips on metal-coated substrates significantly enhances fluorescent signals upon binding of labeled target molecules. This observation holds true for both oligonucleotide and protein microchips. When Cy5 is used as fluorophore, this enhancement is 8-10-fold in hemispherical gel elements and 4-5-fold in flattened gel pads, as compared with similar microchips manufactured on uncoated glass slides. The effect also depends on the hydrophobicity of metal-coated substrate and on the presence of a layer of liquid over the gel pads. The extent of enhancement is insensitive to the nature of formed complexes and immobilized probes and remains linear within a wide range of fluorescence intensities. Manufacturing of gel-based protein microarrays on metal-coated substrates improves their sensitivity using the same incubation time for immunoassay. Sandwich immunoassay using these microchips allows shortening the incubation time without loss of sensitivity. Unlike microchips with probes immobilized directly on a surface, for which the plasmon mechanism is considered responsible for metal-enhanced fluorescence, the enhancement effect observed using hydrogel-based microchips on metal-coated substrates might be explained within the framework of geometric optics.

Development of a biochip with an internal calibration curve for quantitating two forms of the prostate-specific antigen

Three-dimensional gel-based biological microchips were developed for simultaneous quantitation of total (PSAtot) and free (PSAfree) forms of the prostate-specific antigen in human serum in the “one patient, one biochip” format. A method not demanding construction of calibration curves prior to the assay was applied to quantitation of PSAtot and PSAfree. In addition to gel elements with immobilized antibodies against PSAtot and PSAfree, the biochip contains elements with immobilized PSA at different concentrations, forming an internal calibration curve. Data are processed and interpreted with the special-purpose ImaGelAssay program. The sensitivity of the assay is 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. The variation coefficient for measurements with one biochip series does not exceed 10%. The correlation coefficients between the estimates obtained for human sera by the biochip assay and by conventional ELISA were 0.988 for PSAtot and 0.987 for PSAfree.

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to there solution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Array sand MALDI Mass Spectrometry.

Differential quantification of SCCA1 and SCCA2 cancer antigens using a hydrogel biochip

Methods employing hydrogel-based microarrays (biochips) allow the simultaneous monitoring of protein interactions with different antibodies immobilized in gel elements. The method was applied for the simultaneous differential quantification of two highly homologous antigens of squamous cell carcinomas (SCCs) SCCA1 and SCCA2 in a single analysis. Two panels of monoclonal antibodies against recombinant SCCA1 and SCCA2 were generated, and two antibodies, C5 (anti-SCCA1) and A11 (anti-SCCA2), were selected for further evaluation based on their ability to specifically interact with their cognate antigens. Using a sandwich analysis, these antibodies were further tested in combination with anti-SCCA antibodies (H31 and SCC107) recognizing both of the SCCA antigens, thus allowing a quantitative independent measurement of both antigens. The intra- and inter-assay coefficients of variation for all resultant tests did not exceed 10% for the range of SCCA concentrations tested and were independent of whether SCCA1 and SCCA2 concentrations were determined simultaneously. The lower limit of detection (LOD) was estimated as 0.006 ng ml−1 for SCCA1 and 0.011 ng ml−1 for SCCA2 using the SCC107-Cy5 developing antibody and 0.014 ng ml−1 and 0.01 ng ml−1 concentrations, respectively, of the H31-Cy5 developing antibody. This assay provides a simple and accurate procedure for the differential quantitation of SCCA1 and SCCA2 using a single analysis of human serum on a biochip.

Correction: Differential quantification of SCCA1 and SCCA2 cancer antigens using a hydrogel biochip

Correction for ‘Differential quantification of SCCA1 and SCCA2 cancer antigens using a hydrogel biochip’ by Aleksei A. Tikhonov et al., Anal. Methods, 2016, DOI: 10.1039/c6ay02216b.