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Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor.

ABSTRACT: One-step biosensing methods enable the quick and simplified detection of biological substances. In this study, we developed a sensitive one-step method on the basis of a waveguide-mode sensor, which is an optical sensor utilizing waveguide-mode resonance and evanescent light. Streptavidin-conjugated and gold-nanoparticle-conjugated antibodies were reacted with a target substance and applied onto a biotinylated sensing plate. The target substance was detected by observing changes in sensor signals caused by binding the immunocomplex to the sensing surface. Performance of the developed one-step method was examined using a C-reactive protein (CRP) as a target substance. A sensor signal corresponding to the concentration of CRP was obtained. The minimal detectable CRP concentration of the developed method was 10 pM. The developed method greatly simplifies quantitative protein detection without reducing sensitivity.

SUBMITTER: Ashiba H

PROVIDER: S-EPMC7309159 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor.

Ashiba Hiroki H Oyamada Chiaki C Hosokawa Kazuya K Ueno Koji K Fujimaki Makoto M

Sensors (Basel, Switzerland) 20200604 11

One-step biosensing methods enable the quick and simplified detection of biological substances. In this study, we developed a sensitive one-step method on the basis of a waveguide-mode sensor, which is an optical sensor utilizing waveguide-mode resonance and evanescent light. Streptavidin-conjugated and gold-nanoparticle-conjugated antibodies were reacted with a target substance and applied onto a biotinylated sensing plate. The target substance was detected by observing changes in sensor signal ...[more]

PMID: 32512921

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Project description:African swine fever (ASF) is one of the most serious transnational swine diseases in the world. The case fatality rate of susceptible pigs is up to 100%. Currently, no commercial vaccine is available, so the prevention and control of ASF mainly relies on early diagnosis and culling of infected pigs. As the ASF virus continues to evolve, develop, and diversify, nucleic acid testing becomes less efficient. Here, we developed a method for the rapid and direct optical measurement of African swine fever virus (ASFV) antibody in vitro. This one-step procedure requires nearly no sample preparation and involves p30 protein-specific label-free integration into standard 96-well plates. Using a nanoplasmonic biosensor with extraordinary optical transmission (EOT) effect, one-step sample addition, ASFV antibody was detected within 20 min. The positive antibody showed a satisfactory sensitivity and linear relationship in the dilution ratio of 1:100-1:16000. It was used for the detection of clinical serum samples with a coincidence rate of 96.6%. The measurement results can be automatically analyzed and displayed on a conventional microplate meter computer and connected device. Our detection method can be widely applied in point-of-care testing (POCT) of ASFV antibody in pig farms. IMPORTANCE African swine fever (ASF) is a serious transnational disease caused by the African swine fever virus (ASFV), which is highly contagious in wild boars and domestic pigs. There is currently no available vaccine for ASF; therefore, development efforts are a key priority as ASFV continues to evolve and diversify. The ASF antibody rapid detection platform comprising the nanoplasmonic biosensor with extraordinary optical transmission effect can greatly reduce the detection time and improve detection flux while maintaining detection sensitivity and specificity. The one-step sample addition can effectively avoid cross contamination of samples in the detection process. The detection method provides a solution for the rapid and accurate real-time monitoring of ASF in pig farms.

Project description:IntroductionMutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing.ResultsThe IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results.ConclusionsThe new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Dataset Information

Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor.

Publications

Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor.

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