Selection of G-rich ssDNA aptamers for the detection of enterotoxins of the cholera toxin family.

Cholera and milder diarrheal disease are caused by Vibrio cholerae and enterotoxigenic Escherichia coli and are still a prominent public health concern. Evaluation of suspicious isolates is essential for the rapid containment of acute diarrhea outbreaks or prevention of epidemic cholera. Existing detection techniques require expensive equipment, trained personnel and are time-consuming. Antibody-based methods are also available, but cost and stability issues can limit their applications for point-of-care testing. This study focused on the selection of single stranded DNA aptamers as simpler, more stable and more cost-effective alternatives to antibodies for the co-detection of AB5 toxins secreted by enterobacteria causing acute diarrheal infections. Cholera toxin and Escherichia coli heat-labile enterotoxin, the key toxigenicity biomarkers of these bacteria, were immobilized on magnetic beads and were used in a SELEX-based selection strategy. This led to the enrichment of sequences with a high % GC content and a dominant G-rich motif as revealed by Next Generation Sequencing. Enriched sequences were confirmed to fold into G-quadruplex structures and the binding of one of the most abundant candidates to the two enterotoxins was confirmed. Ongoing work is focused on the development of monitoring tools for potential environmental surveillance of epidemic cholera and milder diarrheal disease.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Low-Cost Platform for Multiplexed Electrochemical Melting Curve Analysis.

Detection and identification of single nucleotide polymorphisms (SNPs) have garnered increasing interest in the past decade, finding potential application in detection of antibiotic resistance, advanced forensic science, as well as clinical diagnostics and prognostics, moving toward the realization of personalized medicine. Many different techniques have been developed for genotyping SNPs, and ideally these techniques should be rapid, easy-to-use, cost-effective, flexible, scalable, easily automated, and requiring minimal end-user intervention. While high-resolution melting curve analysis has been widely used for the detection of SNPs, fluorescence detection does not meet many of the desired requirements, and electrochemical detection is an attractive alternative due to its high sensitivity, simplicity, cost-effectiveness, and compatibility with microfabrication. Herein, we describe the multiplexed electrochemical melting curve analysis of duplex surfaces tethered to electrodes of an array. In this approach, thiolated probes designed to hybridize to a DNA sequence containing the SNP to be interrogated are immobilized on gold electrodes. Asymmetric PCR using a ferrocene-labeled forward primer is used to generate this single-stranded redox-labeled PCR amplicon. Following hybridization with the probe immobilized on the electrode surface, the electrode array is exposed to a controlled ramping of temperature, with concomitant constant washing of the electrode array surface while simultaneously carrying out voltammetric measurements. The optimum position of the site complementary to the SNP site in the immobilized probe to achieve maximum differentiation in melting temperature between wild-type and single base mismatch, thus facilitating allelic discrimination, was determined and applied to the detection of a cardiomyopathy associated SNP.

A biophotoelectrode based on boronic acid-modified Chlorella vulgaris cells integrated within a redox polymer.

Green microalgae are gaining attention in the renewable energy field due to their ability to convert light into energy in biophotovoltaic (BPV) cells. The poor exogenous electron transfer kinetics of such microorganisms requires the use of redox mediators to improve the performance of related biodevices. Redox polymers are advantageous in the development of subcellular-based BPV devices by providing an improved electron transfer while simultaneously serving as immobilization matrix. However, these surface-confined redox mediators have been rarely used in microorganism-based BPVs. Since electron transfer relies on the proximity between cells and the redox centres at the polymer matrix, the development of molecularly tailored surfaces is of great significance to fabricate more efficient BPV cells. We propose a bioanode integrating Chlorella vulgaris embedded in an Os complex-modified redox polymer. Chlorella vulgaris cells are functionalized with 3-aminophenylboronic acid that exhibits high affinity to saccharides in the cell wall as a basis for an improved integration with the redox polymer. Maximum photocurrents of (5 ± 1) µA cm-2 are achieved. The developed bioanode is further coupled to a bilirubin oxidase-based biocathode for a proof-of-concept BPV cell. The obtained results encourage the optimization of electron-transfer pathways toward the development of advanced microalgae-based biophotovoltaic devices.

Aptasensors for mycotoxin detection: A review.

Mycotoxins are toxic compounds produced by fungi, which represent a risk to the food and feed supply chain, having an impact on health and economies. A high percentage of feed samples have been reported to be contaminated with more than one type of mycotoxin. Systematic, cost-effective and simple tools for testing are critical to achieve a rapid and accurate screening of food and feed quality. In this review, we describe the various aptamers that have been selected against mycotoxins and their incorporation into optical and electrochemical aptasensors, outlining the strategies exploited, highlighting the advantages and disadvantages of each approach. The review also discusses the different materials used and the immobilization methods employed, with the aim of achieving the highest sensitivity and selectivity.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Hybrid Antibody-Aptamer Assay for Detection of Tetrodotoxin in Pufferfish.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody-aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

A Highly Sensitive Modified Glassy Carbon Electrode with a Carboxylated Multi-walled Carbon Nanotubes/Nafion Nano Composite for Voltammetric Sensing of Dianabol in Biological Fluid.

The extraordinary prerequisite for the analysis of an anabolic steroid, namely dianabol (DB), has inspired towards the development of a cost-effective and high-performance sensing probe. Thus, a simple and robust electrochemical sensor (c-MWCNTs-Nafion®lGCE) for dianabol (DB), a widely used steroid, was developed using a glassy carbon electrode (GCE) modified with functionalized carboxylated multi-walled carbon nanotubes (c-MWCNT) and Nafion®. At pH 7 - 8, differential pulse-cathodic stripping voltammetry (DP-CSV) displayed two cathodic peaks at -0.85 and -1.35 V that varied linearly over a wide range (9.0 × 10-9 (2.7 µg L-1) - 9.0 × 10-6 (2.7 × 103 µg L-1) mol L-1) and 2.9 × 10-6 (8.7 × 102 µg L-1) - 8.0 × 10-5 (2.4 × 104 µg L-1) mol L-1) of DB concentrations, respectively. The low limits of detection and quantification at peak I (-0.85 V) were 2.7 × 10-9 (8.1 × 10-1 ng mL-1) and 9.0 × 10-9 (2.7 ng mL-1) mol L-1, respectively. The repeatability and reproducibility displayed relative standard deviations lower than 5%. The method was applied for DB analysis in human urine and subsequently compared with the standard HPLC method. Interference of common metabolites in biological fluids samples to DB sensing was insignificant. This method has distinctive advantages e.g. precise, short analytical time, sensitive, economical, reproducible and miniaturized sample preparation for DB analysis in biological samples of human origin.

Gold nanoparticle aptamer assay for the determination of histamine in foodstuffs.

The development of a gold nanoparticle aptamer assay is persued for rapid and sensitive determination of histamine in foodstuffs, which could be deployed for on-site use. The assay is based on a histamine-specific aptamer and gold nanoparticles and the salt-induced aggregation of the particles in the presence of histamine indicated by the color change from red to blue. Gold nanoparticle size, salt type, and concentration as well as aptamer concentration were optimized, and using optimum conditions, a limit of detection of 8 nM (~ 0.05 mg/kg) was obtained. Finally, the aptamer AuNP assay was applied to the determination of histamine in quality control fish samples. The histamine levels of these samples had previously been determined using HPLC and commercial ELISA kits by numerous independent laboratories and a good correlation was obtained. The developed AuNP assay is rapid, sensitive, and reproducible. Graphical abstract.

Pediatric glioblastoma target-specific efficient delivery of gemcitabine across the blood-brain barrier via carbon nitride dots.

Pediatric glioblastomas are known to be one of the most dangerous and life-threatening cancers among many others regardless of the low number of cases reported. The major obstacles in the treatment of these tumors can be identified as the lack of prognosis data and the therapeutic requirement to be able to cross the blood-brain barrier (BBB). Due to this lack of data and techniques, pediatric patients could face drastic side effects over a long-time span even after survival. Therefore, in this study, the capability of non-toxic carbon nitride dots (CNDs) to selectively target pediatric glioblastoma cells was studied in vitro. Furthermore, the nanocarrier capability and efficiency of CNDs were also investigated through conjugation of a chemotherapeutic agent and transferrin (Tf) protein. Gemcitabine (GM) was introduced into the system as a chemotherapeutic agent, which has never been successfully used for the treatment of any central nervous system (CNS) cancer. More than 95% of selective damage of SJGBM2 glioma cells was observed at 1 µM of CN-GM conjugate with almost 100% viability of non-cancerous HEK293 cells, although this ability was diminished at lower concentrations. However, further conjugation of Tf to obtain CN-GM-Tf allowed the achievement of selective targeting and prominent anti-cancer activity at a 100-fold lower concentration of 10 nM. Furthermore, both conjugates were capable of effectively damaging several other brain tumor cells, which were not well responsive towards the single treatment of GM. The capability of BBB penetration of the conjugates was observed using a zebrafish model, which confirms the CNDs' competence as an excellent nanocarrier to the CNS.

Yersinia pestis detection using biotinylated dNTPs for signal enhancement in lateral flow assays.

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

Tyrosinase enzyme Langmuir monolayer: Surface chemistry and spectroscopic study.

This study investigates the surface chemistry properties of the tyrosinase enzyme Langmuir monolayer at air-aqueous interface using sodium chloride in the subphase to induce the surface activity of the enzyme. Investigation of surface packing and stability of the tyrosinase Langmuir monolayer were performed using surface chemistry experiments while spectroscopic analysis was done to study enzyme conformation. It was found that the tyrosinase enzyme forms a fluid film at air-aqueous interface with good stability as shown by compression-decompression cycles experiments and stability measurements at various surface pressures. UV-vis absorption and fluorescence measurements at different surface pressures revealed that the Langmuir monolayer has good homogeneity with no evidence of aggregates during compression. To gain insight on the conformation of tyrosinase Langmuir monolayer p-polarized infrared-reflection absorption spectroscopy was used. It was found that at high surface pressures the predominant secondary structures were ß-sheets while at lower surface pressure both α -helices and ß-sheets were present. The circular dichroism spectra were obtained by transferring the Langmuir monolayer at 10 mN.m-1 to a solid quartz support (Langmuir-Blodgett film, LB film), which showed that the major conformation present were α-helices. Images from the immobilized LB films were obtained using atomic force microscopy which showed homogenous and regular deposition with a mean thickness ranging from 3 to 4 nm.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

Nontoxic amphiphilic carbon dots as promising drug nanocarriers across the blood-brain barrier and inhibitors of ß-amyloid.

The blood-brain barrier (BBB) is a main obstacle for drug delivery targeting the central nervous system (CNS) and treating Alzheimer's disease (AD). In order to enhance the efficiency of drug delivery without harming the BBB integrity, nanoparticle-mediated drug delivery has become a popular therapeutic strategy. Carbon dots (CDs) are one of the most promising and novel nanocarriers. In this study, amphiphilic yellow-emissive CDs (Y-CDs) were synthesized with an ultrasonication-mediated methodology using citric acid and o-phenylenediamine with a size of 3 nm that emit an excitation-independent yellow photoluminescence (PL). The content of primary amine and carboxyl groups on CDs was measured as 6.12 × 10-5 and 8.13 × 10-3 mmol mg-1, respectively, indicating the potential for small-molecule drug loading through bioconjugation. Confocal image analyses revealed that Y-CDs crossed the BBB of 5-day old wild-type zebrafish, most probably by passive diffusion due to the amphiphilicity of Y-CDs. And the amphiphilicity and BBB penetration ability didn't change when Y-CDs were coated with different hydrophilic molecules. Furthermore, Y-CDs were observed to enter cells to inhibit the overexpression of human amyloid precursor protein (APP) and ß-amyloid (Aß) which is a major factor responsible for AD pathology. Therefore, data suggest that Y-CDs have a great potential as nontoxic nanocarriers for drug delivery towards the CNS as well as a promising inhibiting agent of Aß-related pathology of the AD.

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Duplex PCR-ELONA for the detection of pork adulteration in meat products.

In this work, a duplex PCR-Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71-188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5-1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products.

Duplex Lateral Flow Assay for the Simultaneous Detection of Yersinia pestis and Francisella tularensis.

High-risk pathogens such as Francisella tularensis and Yersinia pestis are categorized as highly hazardous organisms that can be used as biological weapons. Given the extreme infectivity of these potential biowarfare agents, a rapid, sensitive, cost-effective, and specific method for their detection is required. Here, we report the multiplexed amplification detection of genomic DNA from Francisella tularensis and Yersinia pestis. Amplification was achieved using isothermal recombinase polymerase amplification, exploiting tailed primers, followed by detection using a nucleic-acid lateral flow assay. Excess primers were removed using a novel fishing strategy, avoiding the use of postamplification purification that requires centrifugation and infers additional assay cost. The entire assay is completed in less than 1 h, achieving limits of detection of 243 fg (1.21 × 102 genome equivalent) and 4 fg (0.85 genome equivalent) for Francisella tularensis and Yersinia pestis, respectively.

Carbon dots and gold nanoparticles based immunoassay for detection of alpha-L-fucosidase.

Hepatocellular carcinoma (HCC) is among the leading causes of mortality in the world. The detection of HCC in its early stage is the key for early treatment and thus the improvement of the chances of survival. Among the various methods of HCC screening, assays based on the detection of biomarker that is specific to HCC such as alpha-l-fucosidase (AFU) have been regarded as the most prominent methods. In this regards, a new assay for the detection of AFU to screen HCC was developed. This assay was based on the energy transfer between carbon dots (C-dots) and gold nanoparticles (AuNPs), the concentration of AFU could be monitored by the degree of C-dots fluorescence quenching due to the energy transfer. With this assay, a limit of detection of 3.4 nM (well below the diagnostic cutoff point of 80 nM), and a broad linear range of detection from 11.3 to 200 nM were achieved. We also demonstrate the determination of the concentration of AFU in human blood serum.

Aptamer Selection against a Trichomonas vaginalis Adhesion Protein for Diagnostic Applications.

Trichomoniasis, caused by Trichomonas vaginalis, is the leading nonviral sexually transmitted infection worldwide. We report the selection of a DNA aptamer against a T. vaginalis adhesion protein, AP65, using a microtiter plate-based in vitro combinatorial chemistry process termed systematic evolution of ligands by exponential enrichment. The enriched library pool was sequenced by next-generation sequencing, and several aptamer candidates with high affinity and specificity were identified. The aptamer with the highest affinity and specificity had a KD in the low nanomolar range, as confirmed by three different techniques: surface plasmon resonance, enzyme-linked aptamer assay, and biolayer interferometry. The selected aptamer was demonstrated to have a high specificity to the AP65 protein and to T. vaginalis cells with no cross-reactivity to other enteric and urogenital microorganisms. Current work is focused on the development of inexpensive and easy-to-use aptamer-based diagnostic assays for the reliable and rapid detection of T. vaginalis in vaginal swabs.

Quantification of Nucleic Acid Concentration in the Nanoparticle or Polymer Conjugates Using Circular Dichroism Spectroscopy.

The interface of nucleic acids and nanomaterials is among the most promising fields in recent years. Considerable efforts have been devoted to the development of novel systems based on the two components for various promising applications such as sensing, bioimaging, drug delivery, and theranostics. However, the determination of nucleic acid concentration in these systems remains as a challenge due to the interference of nanoparticles. To this end, we developed a simple, yet reliable, method to quantify the nucleic acid concentration in their nanoparticle or polymer conjugates based on circular dichroism (CD) spectroscopy. In this paper, three nucleic acids, namely, DNA sodium salt from calf thymus (NaDNA), DNA from herring sperm (hsDNA), and ribonucleic acid from torula yeast (tyRNA), were noncovalently conjugated to three nanoparticles. The concentrations of the three nucleic acids in their nanoparticle conjugates were successfully determined on the basis of CD spectra calibration curves.