Biosensor Assay Research Articles

Electrogenerated chemiluminescence biosensor for assay of matrix metalloproteinase-14 and protein-expressing cancer cells via inhibitory peptides-based sandwich assay

A novel electrogenerated chemiluminescence (ECL) biosensor based on sandwich structure between target and inhibitory peptides was firstly developed for determination of matrix metalloproteinase and its expressing cancer cells. Here, target MMP-14 was used as model protein. Inhibitory peptide (AP1) was used as capture probe. Ruthenium complex (Ru) tagged inhibitory peptide (AP2) was used as signal probe (Ru-AP2) in this work. The biosensors were fabricated by covalently coupling Cys group of AP1 onto the surface of functional fullerene-chitosan (C60-Chit) nanocomposite modified glassy carbon electrode. Upon binding of MMP-14, Ru-AP2 further specially bound to MMP-14 and a sandwich format formed, and increasing ECL signal was obtained. Under the optimal experimental conditions, the designed biosensor exhibited excellent sensitivity with a wide linear range of 0.05–7 pg mL−1 and a detection limit of 0.008 pg mL−1. The biosensor is demonstrated by detecting of living cells expressing MMPs (MDA-MB-231 cancer cells as model) and distinguish the expression MMPs in different cells. Our work firstly fabricated a sandwich ECL biosensor for determination of MMPs and MMPs relative cancer cells based on the interaction between peptide inhibitors and PEX of MMPs, which will provide a platform to study the mechanism of MMP and inhibitors.

Development and Application of a Chemical Labeling‐Based Biosensing Assay for Rapid Detection of 8‐Oxoguanine and Its Repair in vitro and in Human Cells

Development and Application of a Chemical <scp>Labeling‐Based</scp> Biosensing Assay for Rapid Detection of <scp>8‐Oxoguanine</scp> and Its Repair <i>in vitro</i> and in Human Cells

Recent Advancements in Electrochemical Biosensors for Monitoring the Water Quality.

The release of chemicals and microorganisms from various sources, such as industry, agriculture, animal farming, wastewater treatment plants, and flooding, into water systems have caused water pollution in several parts of our world, endangering aquatic ecosystems and individual health. World Health Organization (WHO) has introduced strict standards for the maximum concentration limits for nutrients and chemicals in drinking water, surface water, and groundwater. It is crucial to have rapid, sensitive, and reliable analytical detection systems to monitor the pollution level regularly and meet the standard limit. Electrochemical biosensors are advantageous analytical devices or tools that convert a bio-signal by biorecognition elements into a significant electrical response. Thanks to the micro/nano fabrication techniques, electrochemical biosensors for sensitive, continuous, and real-time detection have attracted increasing attention among researchers and users worldwide. These devices take advantage of easy operation, portability, and rapid response. They can also be miniaturized, have a long-life span and a quick response time, and possess high sensitivity and selectivity and can be considered as portable biosensing assays. They are of special importance due to their great advantages such as affordability, simplicity, portability, and ability to detect at on-site. This review paper is concerned with the basic concepts of electrochemical biosensors and their applications in various water quality monitoring, such as inorganic chemicals, nutrients, microorganisms’ pollution, and organic pollutants, especially for developing real-time/online detection systems. The basic concepts of electrochemical biosensors, different surface modification techniques, bio-recognition elements (BRE), detection methods, and specific real-time water quality monitoring applications are reviewed thoroughly in this article.

Ecotoxicity assessment of hydrophobised soils

Hydrophobised soils are a new geomaterial to be used in hydraulic barriers. Insufficient knowledge of their potential harm and environmental risks is a primary hindrance in their application as a fill material. To address this environmental concern, leachate tests and seed germination tests were conducted on two soils (Fujian sand and completely decomposed granite) induced by two hydrophobising compounds (tung oil and dimethyldichlorosilane). Biological indicators – that is, soil respiration, enzyme assays and biosensor assays – were conducted on the hydrophobising agent, tung oil. The total concentrations of heavy metals measured by leachate tests remained below guideline values, indicating that leaching hazard will be negligible during their service life. Biological assays revealed minimal impact on soil microorganisms by tung oil. The results demonstrate negligible environmental impact of hydrophobised soils as fill materials.

Electrochemical sensor for ultrasensitive detection of paraquat based on metal-organic frameworks and para-sulfonatocalix[4]arene-AuNPs composite

The widespread occurrence of pesticides in surface water, groundwater, soil, and food has received increasing attention towards environmental safety. Paraquat (PQ) is world widely used as a rapid sterilant herbicide and is highly toxic to humans. A simple, rapid, sensitive, and on-site detection method for the water environment to detection of PQ is urgently required. Here, we prepared a zeolite imidazole skeleton-8 (ZIF-8) and para-sulfonylcalix[4]arene (pSC4) coated gold nanoparticles composite (pSC4-AuNPs@ZIF-8) by one-step method. An electrochemical biosensor assay for PQ was established based on pSC4-AuNPs@ZIF-8 modified glassy carbon electrode through host-guest recognition of PQ and pSC4. Under the optimal conditions, recoveries of targets determination results were 92.7%–103% (n = 3), respectively. The quantity PQ detection limit was found to be 0.49 pM. Therefore, the signal amplification strategy based on pSC4-AuNPs@ZIF-8 has potential value in detecting trace pollutants in the water environment.

Simultaneous detection of circulating tumor DNAs using a SERS-based lateral flow assay biosensor for point-of-care diagnostics of head and neck cancer.

Circulating tumor DNA (ctDNA) has recently emerged as an ideal target for biomarker analytes. Thus, the development of rapid and ultrasensitive ctDNA detection methods is essential. In this study, a high-throughput surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip is proposed. The aim of this method is to achieve accurate quantification of TP53 and PIK3CA E545K, two types of ctDNAs associated with head and neck squamous cell carcinoma (HNSCC), particularly for point-of-care testing (POCT). Raman reporters and hairpin DNAs are used to functionalize the Pd-Au core-shell nanorods (Pd-AuNRs), which serve as the SERS probes. During the detection process, the existence of targets could open the hairpins on the surface of Pd-AuNRs and trigger the first step of catalytic hairpin assembly (CHA) amplification. The next stage of CHA amplification is initiated by the hairpins prefixed on the test lines, generating numerous "hot spots" to enhance the SERS signal significantly. By the combination of high-performing SERS probes and a target-specific signal amplification strategy, TP53 and PIK3CA E545K are directly quantified in the range of 100 aM-1 nM, with the respective limits of detection (LOD) calculated as 33.1 aM and 20.0 aM in the PBS buffer and 37.8 aM and 23.1 aM in human serum, which are significantly lower than for traditional colorimetric LFA methods. The entire detection process is completed within 45 min, and the multichannel design realizes the parallel detection of multiple groups of samples. Moreover, the analytical performance is validated, including reproducibility, uniformity, and specificity. Finally, the SERS-LFA biosensor is employed to analyze the expression levels of TP53 and PIK3CA E545K in the serum of patients with HNSCC. The results are verified as consistent with those of qRT-PCR. Thus, the SERS-LFA biosensor can be considered as a noninvasive liquid biopsy assay for clinical cancer diagnosis.

Rapid and quantitative detection of tear MMP-9 for dry eye patients using a novel silicon nanowire-based biosensor

Dry eye disease (DED) is the most common chronic eye disease characterized by ocular surface inflammation that affects hundreds of millions of people worldwide. The diagnosis and monitoring of DED require fast and reliable tools in the clinical setting. Matrix metalloproteinase 9 (MMP-9) has been proven to be a reliable indicator of DED owing to its close relationship with inflammation. A novel biosensor based on silicon nanowire-based field-effect transistor (SiNW FET) devices was fabricated for the quantitative measurement of MMP-9 in human tears. A modified controllable process was applied to improve the uniformity of the SiNWs in size and stabilize their performance with optical calibration at low salt concentrations for clinical application. With this protocol, correlation analysis proved the high agreement between the biosensor and enzyme-linked immunosorbent assay (correlation coefficient of 0.92 for DED patients and 0.90 for healthy controls). A diagnostic sensitivity of 86.96% and specificity of 90% were achieved in human tear samples from DED patients and healthy subjects in real-world clinical settings. Furthermore, the tear MMP-9 concentrations monitored using the device correlated with the therapeutic response of the patients with DED. Our enhanced SiNW biosensor device demonstrated its potential as an alternative tool for real-time diagnosis and monitoring for prognostic prediction toward point-of-care testing for DED.

Electrochemical Sensing Platform Based on The Modified Carbon-Ceramic Electrode Using Multiwalled Carbon Nanotubes (MWCNTs) Through The Sol-Gel Process: Application in Sensitive Determination of Morin as A Common Flavonoids

Morin is one of the most common flavonoids which found in nature, plants and vegetable-derived foods and Brazilian yellow tree is a source for its extraction. Because of hydroxyl group existence, Morin represents better antioxidant properties and have great significance in bio electrochemistry-based investigations. In this research work, an electrochemical-based sensing platform was designed based on a modified carbon-ceramic electrode (CCE) through a sol-gel process with carbon nanotubes for the investigation of the electrochemical behavior of Morin. Various electrochemical techniques have been used for the analytical investigation of the proposed sensing platform including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and anodic stripping voltammetry (ASV) methods. Furthermore, the morphological investigation was efficiently performed for the demonstration of electrode surface characteristics. The obtained results showed that the anodic peak currents increased linearly in a concentration range of 0.7– 3 µM, where detection limit is equal to 0.27 µM for DPV, and the linear range is considered to be 0.6-4 µM where there is a lower detection limit of 0.3 µM for ASV. Such a biosensing assay can also prove a great potential for the determination of Morin in dry tea.

State-of-the-art progress of metal-organic framework-based electrochemical and optical sensing platforms for determination of bisphenol A as an endocrine disruptor

Considering the low concentration levels of bisphenol compounds present in environmental, food, and biological samples, and the difficulty in analyzing the matrices, the main challenge is with the cleanup and extraction process, as well as developing highly sensitive determination methods. Recent advances in the field of metal-organic frameworks (MOFs) due to their large surface area, low weight, and other extraordinary physical, chemical, and mechanical features have made these porous materials a crucial agent in developing biosensing assays. This review focuses on MOFs across their definition, structural features, various types, synthetic routes, and their significant utilization in sensing assays for bisphenol A (BPA) determination. Additionally, recent improvements in characteristics and physio-chemical features of MOFs and their functional applications in developing electrochemical and optical sensing assays via different recognition elements for detecting BPA are comprehensively discussed. Finally, the existing boundaries of the current advances including future challenges concerning successful construction of sensing approaches by employing functionalized MOFs are addressed.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·μL-1 (4 copies·μL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

Elucidation of the AI-2 communication system in the food-borne pathogen Campylobacter jejuni by whole-cell-based biosensor quantification

The food-borne pathogen Campylobacter jejuni produces autoinducer-2 (AI-2) as an interspecies signalling molecule. AI-2 can trigger enhanced colonisation and biofilm formation, and this poses a serious risk to public health. To date, this communication system of C. jejuni is only partially understood, as detection and quantification of such autoinducer signalling molecules in complex media is hard to achieve. We have developed a whole-cell Vibrioharveyi-based biosensor assay to accurately quantify and follow production of AI-2 by C. jejuni 81–176 in a defined growth medium and in a model food system. Several V. harveyi strains were tested, but the most sensitive bioluminescent response to C. jejuni AI-2 was achieved with V. harveyi MM30, likely due to its ability to self-amplify the response to AI-2. The AI-2 concentrations measured by this biosensor were confirmed using an independent analytical method, HPLC-FLD, which we introduced for Campylobacter analytics for the first time. The AI-2 concentration produced by C. jejuni 81–176 in the model food system was ∼5-fold that in the defined growth medium, at the same cell density. Together with the linear increments in AI-2 concentrations with cell density, this suggests that in C. jejuni, AI-2 represents a metabolic by-product rather than a true quorum-sensing molecule. This biosensor method is highly sensitive, as shown by the reduction in the limit of detection (by a factor of 100) compared to HPLC-FLD, and it enables quantification of AI-2 in complex matrices, such as food, which will help to improve the quality and safety of food production.

Ultra-efficient multiple cross displacement amplification-lateral flow biosensor (MCDA-LFB) for serogroup identification of prevalent Neisseria meningitidis

Meningococcal disease caused by Neisseria meningitidis remains a major global public health concern. Serogroup A, B, C and W135 were the major disease-causing serogroups. It is vital to timely and efficiently detect and differentiate these four serogroups. Herein, we developed multiple cross displacement amplification-lateral flow biosensor (MCDA-LFB) assays targeting ctrA, sacB, siaD, siaD and synG gene respectively for detection and subtyping of four N. meningitidis serogroups. This assay utilizes LFB to detect FITC and biotin-labeled target amplicons produced by MCDA through double antibody sandwich principle, to allow sensitive and specific detection under a constant temperature. The detection limit was as low as 10 fg or 100 fg genomic DNA in pure cultures and 5.5 CFUs or 36 CFUs in spiked cerebrospinal fluid (CSF) specimens, which were overall 100 to 1000-fold more sensitive than conventional PCR. High specificity of these assays was also validated through type strains and clinical isolates, with no cross-reactions. MCDA-LFB testing procedure can be finished within 1 h. In conclusion, the N. meningitidis- and serogroup-MCDA-LFB assays established in this study are simple, rapid and efficient, providing valuable molecular methods for diagnosis and surveillance of meningococcal disease, especially in resource-limited regions and when specimen culture fails.

The development of a colorimetric biosensing assay for the detection of Helicobacter pylori in feces

As Helicobacter pylori (H. pylori) is closely related to the occurrence of gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer, early detection of H. pylori is an urgent need. In this study, oligonucleotide probes conjugated with gold nanoparticles (AuNPs) were used in combination with H. pylori-specific aptamers for the rapid detection of H. pylori in stool samples, which converted the method of detection from proteins to nucleic acids. Therefore, qualitative detection of H. pylori can be achieved by observing color changes through the aggregation (red to purple) or deaggregation (purple to red) of AuNPs, and further quantitative detection can be achieved through UV spectrometry. The detection limit of the colorimetric biosensing method is 25 CFU/mL (S/N = 3), which is favorably comparable to other reported detection methods. Compared with the existing detection methods for H. pylori, this colorimetric biosensing method has no limitations to the test subjects. All these features render the colorimetric biosensing assay a promising method for the clinical field detection of H. pylori.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay.

BackgroundHaemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical.ResultsIn this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection.ConclusionsTaken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-022-02547-5.

A Multianalyte Electrochemical Genosensor for the Detection of High-Risk HPV Genotypes in Oral and Cervical Cancers.

Infection with high-risk human papillomavirus (HPV) is a major risk factor for oral and cervical cancers. Hence, we developed a multianalyte electrochemical DNA biosensor that could be used for both oral and cervical samples to detect the high-risk HPV genotypes 16 and 18. The assay involves the sandwich hybridization of the HPV target to the silica-redox dye reporter probe and capture probe, followed by electrochemical detection. The sensor was found to be highly specific and sensitive, with a detection limit of 22 fM for HPV-16 and 20 fM for HPV-18, between the range of 1 fM and 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 min. Due to its simplicity, rapidity, and high sensitivity, this biosensor could be used as an alternative method for HPV detection in clinical laboratories as well as for epidemiological studies.

A perspective on ToF-SIMS analysis of biosensor interfaces: Controlling and optimizing multi-molecular composition, immobilization through bioprinting, molecular orientation

The biosensor interface is the heart of biosensing devices and point-of-need platforms. Analyte detection relies on biosensor surface modifications with different molecules (specific binding molecules, blocking molecules, etc.), accomplished following multi-step functionalization and assay procedures, but discrimination of these molecules is not provided by biosensor response. Mass fabrication of biosensors integrated on a chip is facilitated by spatially-selective functionalization through printing of probe molecules, but the resulting multi-molecular distributions are not readily available. Assay efficiency is determined by the orientation and conformation of probe molecules on biosensor surface, but they are rarely determined during assay development. Above challenges in biosensor functionalization and assay can be solved by microanalysis and chemical imaging provided by Time-of-Flight Secondary Ion Mass Spectrometry. This perspective article shows that biosensor surface modifications can be compared with ToF-SIMS not only to determine but also to optimize multi-molecular composition, bioprinting-based immobilization and molecular orientation. These features are controlled through relevant surface phenomena of adsorption and desorption or droplet evaporation as revealed by ToF-SIMS data. The paper highlights the advantages of molecular discrimination with ToF-SIMS, such as accurate characterization of molecular composition that cannot be deduced only by biosensor response, verification of applied biofunctionalization protocol efficiency, and assay optimization with respect to probe immobilization conditions. Finally, application of ToF-SIMS imaging to biomolecules printed into microarrays, biosensors’ arrays or separate biosensor’s components on a chip are presented, with the printed domains comparable, wider or narrower than the nominal diameter of the printing pin. We hope that the reviewed experimental developments could be an inspiration for future research as to how TOF-SIMS analysis methods could assist in our understanding of complex phenomena taking place onto a surface.

Chronic carbon black nanoparticles exposure increases lung cancer risk by affecting the cell cycle via circulatory inflammation

As a widely used pure elemental carbon in colloidal particles, carbon black was listed as a group 2B carcinogen by IARC in 2010. The most available mechanism information about carbon black and carcinogenesis are from in vivo or in vitro studies. However, few studies concerned the nanoparticle's real-ambient exposure causing systemic change and further affecting the target organ. Herein, we used an ex vivo biosensor assay to investigate the transcriptome change of primary bronchial epithelial cells after treatment with the plasma from workers with long-term occupational carbon black exposure history. Based on ex vivo biosensor assay and transcriptome sequencing, we found the effect of internal systemic environment on epithelial cells after carbon black exposure was an inflammatory response, which mainly activates cell cycle-related pathways. After exposure to carbon black, the internal systemic environment could activate cancer-related pathways like epithelial-mesenchymal transition, hypoxia, TNF-α signaling via NF-κB. The hub genes in the carbon black group (CDC20 and PLK1) and their correlation with the systemic environment were uncovered by constructing the protein-protein interaction network. Inflammatory cytokines, especially CRP, were strongly correlated with the expression of CDC20 and PLK1. Besides, we also find a strong correlation between CDC20 and cytokinesis-block micronucleus endpoints in peripheral blood (rho = 0.591, P < 0.001). Our results show that long-term carbon black exposure might activate cell cycle-related pathways through circulating inflammation and increase the risk of cancer, while the oxidative stress caused by diesel exhaust particles are mainly related to PAHs exposure. After exposure to carbon black, the systemic environment could activate cancer-related pathways like diesel exhaust particles, increasing the risk of lung cancer. These attempts might provide a further understanding of the indirect effect of chronic occupational inhaled carbon black exposure on pulmonary carcinogenesis.

Circulatory metabolites trigger ex vivo arterial endothelial cell dysfunction in population chronically exposed to diesel exhaust

BackgroundChronic exposure to diesel exhaust has a causal link to cardiovascular diseases in various environmental and occupational settings. Arterial endothelial cell function plays an important role in ensuring proper maintenance of cardiovascular homeostasis and the endothelial cell dysfunction by circulatory inflammation is a hallmark in cardiovascular diseases. Acute exposure to diesel exhaust in controlled exposure studies leads to artery endothelial cells dysfunction in previous study, however the effect of chronic exposure remains unknown.ResultsWe applied an ex vivo endothelial biosensor assay for serum samples from 133 diesel engine testers (DETs) and 126 non-DETs with the aim of identifying evidence of increased risk for cardiovascular diseases. Environmental monitoring suggested that DETs were exposed to high levels of diesel exhaust aerosol (282.3 μg/m3 PM2.5 and 135.2 μg/m3 elemental carbon). Surprisingly, chronic diesel exhaust exposure was associated with a pro-inflammatory phenotype in the ex vivo endothelial cell model, in a dose-dependent manner with CCL5 and VCAM as most affected genes. This dysfunction was not mediated by reduction in circulatory pro-inflammatory factors but significantly associated with a reduction in circulatory metabolites cGMP and an increase in primary DNA damage in leucocyte in a dose-dependent manner, which also explained a large magnitude of association between diesel exhaust exposure and ex vivo endothelial biosensor response. Exogenous cGMP addition experiment further confirmed the induction of ex vivo biosensor gene expressions in endothelial cells treated with physiologically relevant levels of metabolites cGMP.ConclusionSerum-borne bioactivity caused the arterial endothelial cell dysfunction may attribute to the circulatory metabolites based on the ex vivo biosensor assay. The reduced cGMP and increased polycyclic aromatic hydrocarbons metabolites-induced cyto/geno-toxic play important role in the endothelial cell dysfunction of workers chronic exposure to diesel exhaust.

Ability of matrix metalloproteinase-8 biosensor, IFMA, and ELISA immunoassays to differentiate between periodontal health, gingivitis, and periodontitis.

ObjectiveThe aim of this study was to determine the diagnostic utility of an MMP‐8 biosensor assay in differentiating periodontal health from gingivitis and periodontitis and compare it with an established time‐resolved immunofluorescence assay (IFMA) and enzyme‐linked immunosorbent assay (ELISA).BackgroundCurrently available antibody‐based assays display a wide variability in their ability to accurately measure matrix metalloproteinase‐8 (MMP‐8) levels in saliva.MethodsSalivary MMP‐8 levels were analyzed in 189 systemically healthy participants using an antibody‐based biosensor prototype that operates using a surface acoustic wave technology and compared with IFMA and ELISA antibody assays. Participants were categorized into 3 groups: periodontal health (59), gingivitis (63), and periodontitis (67). A sub‐population of participants (n = 20) with periodontitis received periodontal treatment and were monitored for 6 months.ResultsAll the assays demonstrated significantly higher salivary MMP‐8 concentrations in participants with periodontitis versus gingivitis, periodontitis versus health, and gingivitis versus health (all p < .05). The biosensor data demonstrated significant correlations with IFMA (r = .354, p < .001) and ELISA (r = .681, p < .001). Significant reductions in salivary MMP‐8 concentrations were detected by the biosensor (p = .030) and IFMA (p = .002) in participants with periodontitis 6 months after non‐surgical periodontal treatment. IFMA had the best sensitivity (89.2%) for detecting periodontitis and gingivitis versus health and 96.6% for detecting periodontitis versus health and gingivitis. The biosensor had an AUC value of 0.81 and diagnostic accuracy of 74.2% for differentiating periodontitis and gingivitis from health; an AUC value of 0.86 and diagnostic accuracy of 82.8% for periodontitis versus health and gingivitis.ConclusionsThe biosensor, IFMA, and ELISA assays differentiated between periodontal health, gingivitis, and periodontitis based on salivary MMP‐8 levels. Only the biosensor and, particularly, IFMA identified an effect of periodontal treatment in the participants with periodontitis. Our findings support the potential utility of salivary oral fluid aMMP‐8‐based point‐of‐care technology in the future of periodontal diagnostics.

In vitro Selection of High Affinity DNA and RNA Aptamers that Detect Hepatitis C Virus Core Protein of Genotypes 1 to 4 and Inhibit Virus Production in Cell Culture

Hepatitis C virus (HCV) core is a highly conserved and multifunctional protein that forms the viral capsid, making it an attractive target for HCV detection and inhibition. Aptamers are in vitro selected, single-stranded nucleic acids (RNA or ssDNA) with growing applicability in viral diagnostics and therapy. We have carried out DNA and RNA in vitro selection against six different variants of HCV core protein: two versions of the full-length protein of genotype 1, and the hydrophilic domain of genotypes 1 to 4. The aptamer populations obtained were analyzed by means of Ultra-Deep Sequencing (UDS), the most abundant sequences were identified and a number of highly represented sequence motifs were unveiled. Affinity (measured as the dissociation constant, Kd) of the most abundant DNA and RNA aptamers were quantified using Enzyme-Linked OligoNucleotide Assay (ELONA)-based methods. Some aptamers with nanomolar or subnanomolar Kd values (as low as 0.4 nM) were the common outcome of DNA and RNA selections against different HCV core variants. They were tested in sandwich and competitive biosensor assays, reaching a limit of detection for HCV core of 2 pM. Additionally, the two most prevalent and high affinity aptamers were assayed in Huh-7.5 reporter cell lines infected with HCV, where they decreased both the viral progeny titer and the extracellular viral RNA level, while increasing the amount of intracellular viral RNA. Our results suggest that these aptamers inhibit HCV capsid assembly and virion formation, thus making them good candidate molecules for the design of novel therapeutic approaches for hepatitis C.