Electrochemical DNA biosensor for the multianalyte detection of HPV-16 and HPV-18 in oral and cervical lesions

Infection with high-risk human papillomavirus (HPV) is a major risk factor for oral and cervical cancers. In this study, we developed an electrochemical DNA biosensor for detection of HPV-16 and HPV-18, which are the 2 most prevalent genotypes for development of oral and cervical cancers. The assay involves the sandwich hybridization of the HPV target to silica-redox dye reporter probe and capture probe, followed by electrochemical detection. The sensor was found to be highly specific and sensitive, with detection limit of 22 fM for HPV-16 and 20 fM for HPV-18, between the range of 1 fM to 1 µM. Evaluation with oral and cervical samples showed that the biosensor result was consistent with the nested PCR /gel electrophoresis detection. The biosensor assay could be completed within 90 minutes. Due to its simplicity, rapidity and high sensitivity, this biosensor could be used as an alternative method for HPV detection in clinical laboratories. [151 words]

Rapid electrochemical detection of coronavirus SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

Advances in the Detection of Toxic Algae Using Electrochemical Biosensors

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L−1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

Rapid Electrochemical Detection of Coronavirus SARS-CoV-2.

COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/mL of N and S genes, in less than 2 hours. Sensor evaluation with 105 clinical samples, including 40 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

Direct Plasmonic Detection of Circulating RAS Mutated DNA in Colorectal Cancer Patients

By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The PCR-free method we developed is based on an imaging platform and allows the direct detection of ~1 attomolar RAS sequences in plasma with a sandwich hybridization assay using peptide nucleic acids probes. The assay involves a simple pre-analytical procedure that does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 uL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS SNVs are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. Assay performances were then proven in plasma from CRC patients and healthy donors, demonstrating its promising avenue for cancer monitoring.<br>

Direct Plasmonic Detection of Circulating RAS Mutated DNA in Colorectal Cancer Patients

By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The PCR-free method we developed is based on an imaging platform and allows the direct detection of ~1 attomolar RAS sequences in plasma with a sandwich hybridization assay using peptide nucleic acids probes. The assay involves a simple pre-analytical procedure that does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 uL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS SNVs are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. Assay performances were then proven in plasma from CRC patients and healthy donors, demonstrating its promising avenue for cancer monitoring.<br>

Sandwich-Type DNA Micro-Optode Based on Gold–Latex Spheres Label for Reflectance Dengue Virus Detection

A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP–latex spheres were attached to the thiolated reporter probe (rDNA) by Au–thiol binding to functionalize as an optical gold–latex–rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP–PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP–PSA–rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10−21 M to 1.0 × 10−12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10−29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.

Sensitive detection of microRNA using QCM biosensors: sandwich hybridization and signal amplification by TiO2 nanoparticles

A QCM biosensor for the detection of miR-21 has been demonstrated along with sandwich hybridization and TiO2 nanoparticle-based photocatalytic signal amplification technique.

Single particle ICP-MS-based absolute and relative quantification ofE. coliO157 16S rRNA using sandwich hybridization capture

Herein, a novel 16S rRNA detection platform was achieved by combining a sandwich hybridization reaction, a single-molecule magnetic capture, and single particle-inductively coupled plasma mass spectrometry amplification.