Development of Electrochemical Aptasensor for Lung Cancer Diagnostics in Human Blood.
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ABSTRACT:
SUBMITTER: Shabalina AV
PROVIDER: S-EPMC8659852 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The development of an aptamer-based electrochemical sensor for lung cancer detection is presented in this work. A highly specific DNA-aptamer, LC-18, selected to postoperative lung cancer tissues was immobilized onto a gold microelectrode and electrochemical measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The aptamer protein targets were harvested from blood plasma of lung cancer patients by using streptavidin paramagnetic beads and square wave voltammetry of the samples was performed at various concentrations. In order to enhance the sensitivity of the aptasensor, silica-coated iron oxide magnetic beads grafted with hydrophobic C8 and C4 alkyl groups were used in a sandwich detection approach. Addition of hydrophobic beads increased the detection limit by 100 times. The detection limit of the LC-18 aptasensor was enhanced by the beads to 0.023 ng/mL. The formation of the aptamer - protein - bead sandwich on the electrode surface was visualized by electron microcopy. As a result, the electrochemical aptasensor was able to detect cancer-related targets in crude blood plasma of lung cancer patients.
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Project description:Sensitive detection of ochratoxin A (OTA) is significant and essential because OTA may pose risks to human and animal health. Here, we developed an electrochemical aptasensor for OTA analysis using polyamidoamine (PAMAM) dendrimers as a signal amplifier. As a carrier, PAMAM has numerous primary amino groups that can be coupled with thiolated complementary strand DNA (cDNA), allowing it to recognize aptamers bound to the surface of horseradish peroxidase (HRP)-modified gold nanoparticles (AuNPs), thereby improving the sensitivity of the aptasensor. When monitoring the positive samples, OTA was captured by the aptamer fixed on the HRP-conjugated AuNP surface by specific recognition, after which the formed OTA-aptamer conjugates were detached from the electrode surface, ultimately decreasing the electrochemical signal monitored by differential pulse voltammetry. The novel aptasensor achieved a broad linear detection range from 5 to 105 ng L-1 with a low detection limit of 0.31 ng L-1. The proposed aptasensor was successfully applied for OTA analysis in red wine, with recovery rates ranging from 94.15 to 106%. Furthermore, the aptasensor also exhibited good specificity and storage stability. Therefore, the devised aptasensor represents a sensitive, practical and reliable tool for monitoring OTA in agricultural products, which can also be adapted to other mycotoxins.
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Project description:A selection of aptamers specific for di(2-ethylhexyl) phthalate (DEHP) and development of electrochemical impedance spectroscopy (EIS) aptasensor are described in this paper. The aptamers were selected from an immobilized ssDNA library using the systematic evolution of ligands by exponential enrichment (SELEX). The enrichment was monitored using real-time quantitative PCR (Q-PCR), and the aptamers were identified by high-throughput sequencing (HTS), gold nanoparticles (AuNPs) colorimetric assay, and localized surface plasmon resonance (LSPR). The EIS aptasensor was developed to detect DEHP in water samples. After eight rounds of enrichment, HTS, AuNPs colorimetric assay, and LSPR analysis indicated that four aptamers had higher binding activity, and aptamer 31 had the highest affinity (Kd = 2.26 ± 0.06 nM). The EIS aptasensor had a limit of detection (LOD) of 0.103 pg/mL with no cross-reactivity to DEHP analogs and a mean recovery of 76.07% to 141.32% for detection of DEHP in water samples. This aptamer is novel with the highest affinity and sensitivity.
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Project description:Seventh generation Exiqon locked nucleic acid miRCURY LNA microarrays were used to profile the expression of microRNAs in whole blood of patients with lung cancer and of clinically relevant controls without the disease (individuals with non-cancerous lung nodule, at high risk for cancer because of heavy smoking, or with lung cancer treated by surgical resection). The goal was to examine if whole blood microRNAs can be used for developing a non-invasive assay to diagnose lung cancer. Using the PAXgene Blood miRNA kit (Qiagen, Valencia, CA, USA), total RNA was isolated from whole blood stabilized and stored in PAXgene Blood RNA tubes for 85 individuals with lung cancer, 59 individuals without the disease but at a high risk for the disease because of smoking, and 17 individuals with a non-cancerous pulmonary nodule. RNA was also isolated from 13 samples of blood of 12 of the lung cancer cases following surgery; blood samples were collected on two different days post-operatively for one of the cases (Sample names 157 and 160). The data-set here includes data (sample name 171) from a post-operative blood sample of an individual (who had the lung cancer removed by operation) for whom good array data was not obtained for RNA from a pre-operative blood sample. Two RNA samples were used to test technical duplicability of the microarray platform (pairs of sample names 59 & 192, and 175 & 182). Thus, a total of 162 unique individuals and 175 unique RNA samples are covered by this data-set. Isolated RNA was provided to Exiqon (Denmark) for microRNA quantification using microarrays. Quality and concentration of RNA in the samples was assessed by Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) instruments. Sample RNA (500 ng) was labeled with the Cy3-like Hy3 dye, mixed with 500 ng of a human universal reference RNA (product number AM6000, Ambion, Austin, TX, USA) that was labeled with the Cy5-like Hy5 dye, and hybridized to 7th generation miRCURY locked nucleic acid oligonucleotide microarrays. Sixty-two artificial small RNAs were spiked-in each RNA sample before labeling. disease state: Lung cancer (before surgical resection of lung cancer): 1, 2, 4, 5, 6, 7, 8, 9, 10, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 63, 64, 65, 67, 68, 69, 70, 81, 82, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 127, 149, 151, 153, 154, 156, 158, 165, 167, 174, 175, 176, 177, 179, 180, 181, 183, 184, 186, 187, 188, 189, 193 disease state: Non-cancerous lung nodule: 128, 129, 130, 131, 133, 134, 135, 136, 137, 140, 141, 142, 144, 145, 146, 148, 182, 190 disease state: None (after surgical resection of lung cancer): 155, 157, 159, 160, 161, 162, 163, 164, 166, 169, 170, 171, 172, 173 disease state: None (no current or past lung cancer or non-cancerous nodule): 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 113, 114, 115, 116, 117, 118, 119, 120, 192 individual: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162
| S-ECPF-GEOD-40738 | biostudies-other
Project description:Electrochemical and optical platforms are commonly employed in designing biosensors. However, one signal readout can easily lead to inaccuracies due to the effect of nonstandard test procedures, different operators, and experimental environments. We have developed a dual-signal protocol that combined two transducer principles in one aptamer-based biosensor by simultaneously performing electrochemical- and extraordinary optical transmission (EOT)-based plasmonic detection using gold nanopit arrays (AuNpA). Compared with full hole structures, we found that nanopits, that did not fully penetrate the gold film, not only exhibited a better plasmonic bandwidth and refractive index sensitivity both in the finite-difference time-domain simulation and in experiments by shielding the gold/quartz mode but also enlarged the electrochemical active surface area. Therefore, the periodic non-fully penetrating AuNpA were modified with ferrocene-labeled human serum albumin aptamer receptors. The formation of the receptor layer and human serum albumin binding complex induced a conformational change, which resulted in variation in the electron transfer between the electro-active ferrocene units and the AuNpA surface. Simultaneously, the binding event caused a surface plasmon polaritons wavelength shift corresponding to a change in the surface refractive index. Interestingly, although both transducers recorded the same binding process, they led to different limits of detection, dynamic ranges, and sensitivities. The electrochemical transducer showed a dynamic detection range from 1 nM to 600 μM, while the optical transducer covered high concentrations from 100 μM to 600 μM. This study not only provides new insights into the design of plasmonic nanostructures but also potentially opens an exciting avenue for dual-signal disease diagnosis and point-of-care testing applications.
| S-EPMC10458775 | biostudies-literature
Project description:BackgroundTuberculous meningitis (TBM) is the most devastating manifestation of extra-pulmonary tuberculosis. About 33% of TBM patients die due to very late diagnosis of the disease. Conventional diagnostic methods based on signs and symptoms, cerebrospinal fluid (CSF) smear microscopy or liquid culture suffer from either poor sensitivity or long turnaround time (up to 8 weeks). Therefore, in order to manage the disease efficiently, there is an urgent and unmet need for a rapid and reliable diagnostic test.MethodsIn the current study, to address the diagnostic challenge of TBM, a highly rapid and sensitive structural switching electrochemical aptasensor was developed by combining the electrochemical property of methylene blue (MB) with the molecular recognition ability of a ssDNA aptamer. To demonstrate the clinical diagnostic utility of the developed aptasensor, a blinded study was performed on 81 archived CSF specimens using differential pulse voltammetry.ResultsThe electrochemical aptasensor developed in the current study can detect as low as 10 pg HspX in CSF background and yields a highly discriminatory response (P<0.0001) for TBM and not-TBM categories with ~95% sensitivity and ~97.5% specificity and has the ability to deliver sample-to-answer in ≤30 minutes.ConclusionIn summary, we demonstrate a new aptamer-based electrochemical biosensing strategy by exploiting the target-induced structural switching of H63 SL-2 M6 aptamer and electroactivity of aptamer-tagged MB for the detection of HspX in CSF samples for the diagnosis of TBM. Further, the clinical utility of this sensor could be extended for the diagnosis of other forms of tuberculosis in the near future.
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