Paper-based microfluidic chip for rapid detection of SARS-CoV-2 N protein.

This research has developed a method for rapid detection of SARS-CoV-2 N protein on a paper-based microfluidic chip. The chitosan-glutaraldehyde cross-linking method is used to fix the coated antibody, and the sandwich enzyme-linked immunosorbent method is used to achieve the specific detection of the target antigen. The system studied the influence of coating antibody concentration and enzyme-labeled antibody concentration on target antigen detection. According to the average gray value measured under different N protein concentrations, the standard curve of the method was established and the sensitivity was tested, and its linear regression was obtained. The equation is y = 9.8286x+137.6, R2 = 0.9772 > 0.90, which shows a high degree of fit. When the concentration of coating antibody and enzyme-labeled antibody were 1 µg/mL and 2 µg/mL, P > 0.05, the difference was not statistically significant, so the lower concentration of 1 µg/mL was chosen as the coating antibody concentration. The results show that the minimum concentration of N protein that can be detected by this method is 8 µg/mL, and the minimum concentration of coating antibody and enzyme-labeled antibody is 1 µg/mL, which has the characteristics of high sensitivity and good repeatability.

Vortex- and Centrifugation-Free Extraction of HIV-1 RNA.

BACKGROUND AND OBJECTIVE: HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments. METHODS: A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil-water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples. RESULTS: Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%. CONCLUSIONS: From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

Comparison of body temperature readings between an implantable microchip and a cloacal probe in lorikeets (Trichoglossus haematodus sp.).

Body temperature readings can be a useful diagnostic tool for identifying the presence of subclinical disease. Traditionally, rectal or cloacal thermometry has been used to obtain body temperatures. The use of implantable microchips to obtain these temperatures has been studied in a variety of animals, but not yet in avian species. Initially, timepoint one (T1), nine lorikeets were anesthetized via facemask induction with 5% isoflurane and maintained at 2-3% for microchip placement and body temperature data collection. Body temperature was measured at 0 and 2 min post-anesthetic induction both cloacally, using a Cardell veterinary monitor and also via implantable microchip, utilizing a universal scanner. On two more occasions, timepoints two and three (T2, T3), the same nine lorikeets were manually restrained to obtain body temperature readings both cloacally and via microchip, again at minutes 0 and 2. There was no statistical difference between body temperatures, for both methods, at T1. Microchip temperatures were statistically different than cloacal temperatures at T2 and T3. Body temperatures at T1, were statistically different from those obtained at T2 and T3 for both methods. Additional studies are warranted to verify the accuracy of microchip core body temperature readings in avian species.

Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms.

The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples.

Rapid extraction and preservation of genomic DNA from human samples.

Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

Programmable bio-nano-chip systems for serum CA125 quantification: toward ovarian cancer diagnostics at the point-of-care.

Point-of-care (POC) implementation of early detection and screening methodologies for ovarian cancer may enable improved survival rates through early intervention. Current laboratory-confined immunoanalyzers have long turnaround times and are often incompatible with multiplexing and POC implementation. Rapid, sensitive, and multiplexable POC diagnostic platforms compatible with promising early detection approaches for ovarian cancer are needed. To this end, we report the adaptation of the programmable bio-nano-chip (p-BNC), an integrated, microfluidic, and modular (programmable) platform for CA125 serum quantitation, a biomarker prominently implicated in multimodal and multimarker screening approaches. In the p-BNCs, CA125 from diseased sera (Bio) is sequestered and assessed with a fluorescence-based sandwich immunoassay, completed in the nano-nets (Nano) of sensitized agarose microbeads localized in individually addressable wells (Chip), housed in a microfluidic module, capable of integrating multiple sample, reagent and biowaste processing, and handling steps. Antibody pairs that bind to distinct epitopes on CA125 were screened. To permit efficient biomarker sequestration in a three-dimensional microfluidic environment, the p-BNC operating variables (incubation times, flow rates, and reagent concentrations) were tuned to deliver optimal analytical performance under 45 minutes. With short analysis times, competitive analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and limit of detection of 1.0 U/mL) was achieved on this minisensor ensemble. Furthermore, validation with sera of patients with ovarian cancer (n = 20) showed excellent correlation (R(2) = 0.97) with gold-standard ELISA. Building on the integration capabilities of novel microfluidic systems programmed for ovarian cancer, the rapid, precise, and sensitive miniaturized p-BNC system shows strong promise for ovarian cancer diagnostics.

Comparison of temperature readings from a percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer in equids.

OBJECTIVE: To compare temperature readings from an implantable percutaneous thermal sensing microchip with temperature readings from a digital rectal thermometer, to identify factors that affect microchip readings, and to estimate the sensitivity and specificity of the microchip for fever detection. DESIGN: Prospective study. ANIMALS: 52 Welsh pony foals that were 6 to 10 months old and 30 Quarter Horses that were 2 years old. PROCEDURES: Data were collected in summer, winter, and fall in groups 1 (n = 23 ponies), 2 (29 ponies), and 3 (30 Quarter Horses), respectively. Temperature readings from a digital rectal thermometer and a percutaneous thermal sensing microchip as well as ambient temperature were recorded daily for 17, 23, and 19 days in groups 1 through 3, respectively. Effects of ambient temperature and rectal temperature on thermal sensor readings were estimated. Sensitivity and specificity of the thermal sensor for detection of fever (rectal temperature, >or= 38.9 degrees C [102 degrees F]) were estimated separately for data collection at ambient temperatures 15.6 degrees C. RESULTS: Mean ambient temperatures were 29.0 degrees C (84.2 degrees F), -2.7 degrees C (27.1 degrees F), and 10.4 degrees C (50.8 degrees F) for groups 1 through 3, respectively. Thermal sensor readings varied with ambient temperature and rectal temperature. Rectal temperatures ranged from 36.2 degrees to 41.7 degrees C (97.2 degrees to 107 degrees F), whereas thermal sensor temperature readings ranged from 23.9 degrees (75 degrees F) to 42.2 degrees C (75 degrees to 108 degrees F). Sensitivity for fever detection was 87.4%, 53.3%, and 58.3% in groups 1 to 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The thermal sensor appeared to have potential use for initial screening of body temperature in equids at ambient temperatures > 15.6 degrees C.

Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms.

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.