Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

Electrochemical ELASA: improving early cancer detection and monitoring.

The discovery of new molecular biomarkers of cancer during the last decades and the development of new diagnostic devices exploiting those have significantly contributed to the clinical analysis of cancer and to improve the outcomes. Among those, liquid biopsy sensors exploiting aptamers for the detection of cancer biomarkers in body fluids are useful and accurate tools for a fast and inexpensive non-invasive screening of population. The incorporation of aptamers in electrochemical sandwich biosensors using enzyme labels, a so-called ELASA, has demonstrated its utility to improve the detection schemes. In this review, we overview the existing ELASA assays for numerous cancer biomarkers as alternatives to the traditional ELISA and discuss their possibilities to reach the market, currently dominated by optical immunoassays.

Developing a DNA aptamer-based approach for biosensing cystatin-c in serum: An alternative to antibody-based methods.

Oligonucleotide aptamers are short, synthetic and single-stranded DNA or RNA molecules capable of binding to a wide range of molecules, from small molecules to large cells. Nowadays, aptamers are valuable tools in research, clinical diagnosis and treatment. Their small size and high specificity in addition to their lack of immunogenicity make them great alternatives to other diagnosing candidates such as antibodies. In this study, we have introduced a new method based on competitive Enzyme-Linked Aptamer Sorbent Assay (ELASA) using single-stranded DNA (ssDNA) aptamers to measure cystatin-c levels in serum samples. To this aim, through a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process a number of aptamers were selected from which an aptamer with a Kd (dissociation constant) value of 65.5 ±â€¯0.007 nM was chosen for further analyses. The limit of detection (LoD) was found to be 216.077 pg/ml. The results of the analytical application of this method in serum samples were comparable to those of commonly used commercial kits.

Development of novel aptamer-based enzyme-linked apta-sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus.

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)-based enzyme-linked apta-sorbent assay (VA2-ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2-ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2-ELASA could specifically identify V. alginolyticus, but not other non-target bacterial strains. VA2-ELASA could detect V. alginolyticus at the concentration of 5 × 104 /ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2-ELASA in this study. It took less than one hour to accomplish the detection process by VA2-ELASA. The characteristics of specificity, sensitivity and easy operation make VA2-ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2.

Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

Development and characterization of aptamer-based enzyme-linked apta-sorbent assay for the detection of Singapore grouper iridovirus infection.

AIMS: Singapore grouper iridovirus (SGIV) is a devastating aquaculture virus responsible for heavy economic losses to grouper, Epinephelus sp. aquaculture. The aim of this study was to develop a rapid and sensitive detection method for SGIV infections in infected groupers. METHODS AND RESULTS: We previously generated DNA aptamers against SGIV-infected cells. In this study, we established and characterized a novel aptamer (Q3)-based enzyme-linked apta-sorbent assay (ELASA) for the detection of SGIV infection in Epinephelus coioides. The Q3-based ELASA could detect SGIV infection rapidly in vitro and in vivo, with high specificity and stability. Q3-based ELASA specifically recognized SGIV-infected cells, but not other-virus-infected cells or uninfected cells. Q3-based ELASA detected SGIV infection in a dose-dependent manner at Q3 concentrations as low as 125 nmol l(-1) . The results in relation to SGIV-infected cells (5 × 10(4) ), incubation time (1 min) and incubation temperature (37°C) demonstrated that Q3-based ELASA could detect SGIV infection quickly and stably, superior to antibody-based enzyme-linked immunosorbent assay. Q3-based ELASA could detect the presence of SGIV infection in kidney, liver and spleen samples in vivo, at dilutions of 1/50, 1/100 and 1/50 respectively. The complete detection process took 1-2 h. CONCLUSIONS: Q3-based ELASA could be a useful tool for diagnosing SGIV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first developed aptamer-based ELASA for detecting SGIV infection, and is widely applicable in grouper aquaculture industry in light of its rapidity, and high specificity and stability.

Isolation of a novel DNA aptamer against LipL32 as a potential diagnostic agent for the detection of pathogenic Leptospira.

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira and for rapid diagnostics, direct detection is desirable. LipL32 protein is the most suitable biomarker for direct detection. DNA aptamers are sought to be generated against LipL32 by Systemic Evolution of Ligands via Exponential Enrichment (SELEX). LepDapt-5a is the most potent aptamer candidate among all the candidates, as determined by direct Enzyme-linked Aptasorbent Assay (ELASA). LepDapt-5a was predicted to form a G-quadruplex structure as predicted by QGRS Mapper and validated experimentally by direct ELASA. The diagnostic potential of the aptamer was further tested on a direct and sandwich ELASA platform. A LOD of 106 mL-1 and 105 mL-1 were estimated by direct and sandwich ELASA platforms, respectively, which are within the range associated with leptospiremia levels. The dot blot assay developed was able to attain a LOD of 104 CFU mL-1 against pathogenic Leptospira, which is also within the leptospiremia level. This is the first-ever DNA aptamer and hybrid-heterodimeric aptamer constructed against LipL32. The diagnostic potentiality of the LepDapt-5a DNA aptamer was proven on three major diagnostic platforms, which are direct ELASA, sandwich ELASA, and aptamer-based dot assay.

The Diagnostic Potential of RNA Aptamers against the NS1 Protein of Dengue Virus Serotype 2.

Dengue infection, caused by the dengue virus, is a global threat which requires immediate attention and appropriate disease management. The current diagnosis of dengue infection is largely based on viral isolation, RT-PCR and serology-based detection, which are time-consuming and expensive, and require trained personnel. For early diagnosis of dengue, the direct detection of a dengue antigenic target is efficacious, and one such target is NS1. NS1-based detection is primarily antibody-centric and is beset by drawbacks pertaining to antibodies such as the high cost of synthesis and large batch-to-batch variation. Aptamers are potential surrogates of antibodies and are much cheaper, without exhibiting batch-to-batch variation. Given these advantages, we sought to isolate RNA aptamers against the NS1 protein of dengue virus serotype 2. A total of 11 cycles of SELEX were carried out, resulting in two potent aptamers, DENV-3 and DENV-6, with dissociation constant values estimated at 37.57 ± 10.34 nM and 41.40 ± 9.29 nM, respectively. These aptamers can be further miniaturized to TDENV-3 and TDENV-6a with an increased LOD upon their usage in direct ELASA. Moreover, these truncated aptamers are highly specific against the dengue NS1 while showing no cross-reactivity against the NS1 of the Zika virus, the E2 protein of the Chikungunya virus or the LipL32 protein of Leptospira, with target selectivity retained even in human serum. The usage of TDENV-3 as the capturing probe and TDENV-6a as the detection probe underpinned the development of an aptamer-based sandwich ELASA for the detection of dengue NS1. The sensitivity of the sandwich ELASA was further improved with the stabilization of the truncated aptamers and the repeated incubation strategy, which enabled a LOD of 2 nM when used with the target NS1 spiked in human serum diluted at 1:2000.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for Detection of Epsilon Toxin.

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX. Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX. Results: Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 µg/mL for ETX3 and ETX11, respectively. . Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

Aptamer-based diagnosis of various SARS-CoV2 strains isolated from clinical specimens.

The emergence of the SARS-CoV-2 virus, an unknown strain of coronavirus, has resulted in severe acute respiratory syndrome with high mortality rates worldwide. Due to the possibility of asymptomatic carriers, late diagnosis of infected individuals can lead to uncontrollable transmission of the disease, making early and accurate detection crucial in controlling the spread of the virus. In this study we identified high-binding-affinity aptamers targeting various strains of the SARS-CoV2 (COVID-19) virus, using the GO-Cell-SELEX (Graphene Oxide- Systematic Evolution of Ligands by Exponential Enrichment) strategy. A total of 96 aptamers were developed through 11 rounds of GO-Cell-SELEX from a random 40 nucleotide single-strand DNA (ssDNA) aptamer library. Using the surface plasmon resonance (SPR) method, the dissociation constant (Kd) values of all aptamers were calculated and two aptamers 52 and 91 with Kd 50 and 61 were selected for enzyme-linked apta-sorbent assay (ELASA). Aptamer 91 could detect various strains of the virus in above 97% of clinical samples obtained from nasopharyngeal swaps (NPS) specimens kept in viral transport media (VTM), confirmed by real-time PCR assay at COVID-19 Reference Diagnostic Laboratory of Iran, Pasture Institute. Aptamer 52 could detect the SARS-CoV2 virus in a competitive lateral flow assay (LFA) to be considered for a future designed kit. These two simple, specific, and sensitive tests can be used in combination for rapid and early diagnosis of various strains of the COVID-19 virus. Our results suggest that these two discovered aptamers present an opportunity for developing a new rapid aptamer-based coronavirus diagnostic kit.

A Novel Sandwich ELASA Based on Aptamer for Detection of Largemouth Bass Virus (LMBV).

Largemouth bass virus (LMBV) is a major viral pathogen in largemouth bass culture, usually causing high mortality and heavy economic losses. Accurate and early detection of LMBV is crucial for diagnosis and control of the diseases caused by LMBV. Previously, we selected the specific aptamers, LA38 and LA13, targeting LMBV by systematic evolution of ligands by exponential enrichment (SELEX). In this study, we further generated truncated LA38 and LA13 (named as LA38s and LA13s) with high specificity and affinities and developed an aptamer-based sandwich enzyme-linked apta-sorbent assay (ELASA) for LMBV diagnosis. The sandwich ELASA showed high specificity and sensitivity for the LMBV detection, without cross reaction with other viruses. The detection limit of the ELASA was as low as 1.25 × 102 LMBV-infected cells, and the incubation time of the lysate and biotin labeled aptamer was as short as 10 min. The ELASA could still detect LMBV infection in spleen lysates at dilutions of 1/25, with good consistency of qRT-PCR. For the fish samples collected from the field, the sensitivity of ELASA was 13.3% less than PCR, but the ELASA was much more convenient and less time consuming. The procedure of ELASA mainly requires washing and incubation, with completion in approximately 4 h. The sandwich ELASA offers a useful tool to rapidly detect LMBV rapidly, contributing to control and prevention of LMBV infection.

Selection and characterization of structure-switching DNA aptamers for the salivary peptide histatin 3.

In this study, single-stranded DNA aptamers that switch structural conformation upon binding to the salivary peptide histatin 3 have been reported for the first time. Histatin 3 is an antimicrobial peptide that possesses the capability of being a therapeutic agent against oral candidiasis and has recently been linked as a novel biomarker for acute stress. The aptamers were identified through a library immobilization version of an iterative in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment (SELEX). Through the SELEX process, four unique aptamer candidates sharing a consensus sequence were identified. These selected sequences exhibited binding affinity and specificity to histatin 3 and in order to further characterize these aptamers, a direct format enzyme-linked aptamer sorbent assay (ELASA) was developed. The best performing candidate demonstrated an equilibrium dissociation constant (Kd) value of 1.97 ± 0.48 µM. These novel aptamers have the potential to lead to the further development of refined sensing assays and platforms for the detection and quantification of histatin 3 in human saliva and other biological media.

Colorimetric and chemiluminescence based enzyme linked apta-sorbent assay (ELASA) for ochratoxin A detection.

Ochratoxin A (OTA) is one of the most widespread mycotoxin found to contaminate various food products such as cereals, spices, groundnuts, coffee, wine, beer etc. It is also carried over from contaminated feed and fodder to milk, blood, meat, kidney and liver of animals consuming it. Enzyme-linked to biorecognition molecules like antibodies or aptamers are very popular due to their ability to be used as labels or tags in biosensing formats. In this work, OTA aptamer based colorimetric and chemiluminescence biosensing formats were evaluated for the detection of OTA. The colorimetric enzyme linked apta-sorbent assay (Co-ELASA) and chemiluminescence enzyme linked apta-sorbent assay (Cl-ELASA) showed a linear detection range from 1 pg/mL to 1 µg/mL with a limit of detection (LOD) of 0.84 pg/mL for Co-ELASA (limit of quantification (LOQ) = 2.54 pg/mL) and 1.29 pg/mL for Cl-ELASA (LOQ = 3.94 pg/mL) under optimized buffer conditions. Comparison of ELASA methods with sandwich ELISA indicated that the developed techniques had sensitivity similar to the conventional technique which indicated a LOD of 1.13 pg/mL and LOQ of 3.41 pg/mL. Studies in simulated contaminated food samples by spiking OTA in groundnut and coffee bean at concentrations of 0.1, 1 and 10 ppb, indicated recoveries in the range of 50.21 to 113.27% for Co-ELASA, 90.47 to 107.72% for Cl-ELASA and 76.23 to 141.49% for ELISA. Results of the study indicate that Co-ELASA and Cl-ELASA assays could be an alternate approach for ultrasensitive detection of OTA in food samples, which can also be adapted for biosensor development.

Terminal deoxynucleotidyl transferase based signal amplification for enzyme-linked aptamer-sorbent assay of colorectal cancer exosomes.

Exosomes have received increasingly significant attention and have shown great clinical value as biomarkers for a number of diseases. However, there is still a lack of a highly sensitive and visualized method for the detection of exosomes in numerous samples simultaneously. Here, we developed a high-throughput, colorimetric and simple method to detect colorectal cancer (CRC) exosomes based on terminal deoxynucleotidyl transferase (TdT)-aided ultraviolet signal amplification. Anti-A33, a CRC exosomal protein marker, was selected as a capture probe, and a facility-prepared EpCAM (CRC exosomal protein) aptamer-Au-primer complex was used as a signal probe. After the CRC exosomes were captured onto the surface of 96-well plates, the primer was extended to the poly(biotin-adenine) chains with the help of TdT, resulting in an increase in the binding amount of avidin-modified horseradish peroxidase (Av-HRP) for H2O2-mediated oxidation of 3,3',5,5'-tetramethyl benzidine (TMB) in enzyme-linked aptamer-sorbent assay (ELASA). The results showed that the incorporation of ploy(biotin-A) enabled approximately 10.4-fold signal amplification. This approach achieved a linear range of 9.75 × 103-1.95 × 106 particles/µL for CRC cell-derived exosomes. The feasibility of the developed assay was evaluated using clinical serum samples. CRC patients (n = 16) could be clearly and successfully distinguished from healthy individuals (n = 9). Furthermore, this proposed platform holds considerable potential for the detection of different targets, simply by changing the aptamer and antibody.

Highly adaptable and sensitive FRET-based aptamer assay for the detection of Salmonella paratyphi A.

Here we demonstrate a facile and versatile fluorescence resonance energy transfer (FRET) based aptasensor for rapid detection of Salmonella paratyphi A. The assay shows a detection limit up to 10 cfu·mL-1 with no cross-reactivity with other bacterial species. Less than 8% of inter-assay coefficient variance and recovery rate between 85 and 102% attests the assay reliability. The advantages of FRET-based aptamer assay over the conventional immunoassay formats such as ELISA are the specificity, speed, reliability, and simplicity of the assay. The ssDNA aptamers specific towards pathogenic Salmonella paratyphi A were generated via whole-cell SELEX. The aptamer was conjugated onto quantum dot (QD) that served as the molecular beacon and graphene oxide (GO) was used as a fluorescence quencher. Thus the proposed method enables detection of target pathogen using FRET-based assay. Further interaction of aptamer with pathogen protein DNA gyrase was explored using classical molecular dynamics simulation.

Gold nano-urchin integrated label-free amperometric aptasensing human blood clotting factor IX: A prognosticative approach for "Royal disease".

This article is clearly presenting the development of a biosensor for human factor IX (FIX) to diagnose the blood clotting deficiency, a so-called 'Royal disease' using an interdigitated electrode (IDE) with the zinc oxide surface modification. Gold nano-urchins (GNUs) with 60 nm in diameter was integrated into a streptavidin-biotinylated aptamer strategy to enhance the active surface area. Two different comparative studies have been done to validate the system to be practiced in the current work holds with a higher capability for the high-performance sense. Whereby, the presence and absence of GNUs in the aptasensing system for FIX interaction were investigated using the amperometric measurement, using a linear sweep voltage of 0-2 V at 0.01 V step voltage. The detection limit was 6 pM based on 3σ calculation when GNUs integrated aptamer assay was utilized for FIX detection, which shows 8 folds sensitivity enhancement comparing the condition in the absence of GNU and 50 folds higher than sensitive radio-isotope and surface plasmon resonance assays. Albeit, the surface and molecular characterizations were well demonstrated by scanning electron microscopy, atomic force microscopy, 3D nano-profilometry and further supports were rendered by UV-Vis spectroscopy and Enzyme-linked apta-sorbent assay (ELASA). Furthermore, the spiking experiment was done by FIX-spikes in human blood serum in order to demonstrate the stability with a higher non-fouling.

Comparison of Flow Cytometry and ELASA for Screening of Proper Candidate Aptamer in Cell-SELEX Pool.

Aptamers are single-stranded RNA or DNA, which bind to their target with high affinity and specificity. Method of isolating aptamers against cell surface protein is called cell-SELEX. Common approach for monitoring cell-SELEX developed aptamers is flow cytometry. Since flow cytometry is costly and requires sophisticated equipments, we suggested implementing easy access, high throughput enzyme-link apta-sorbent assay test (ELASA) to confirm the specificity of aptamers selected through cell-SELEX process. In this regard, we compared ELASA and flow cytometry techniques in order to screen potent candidate aptamers against A2780 Rcis cell line, which were selected by cell-SELEX. The obtained results demonstrated that both ELASA and flow cytometry are identical in terms of sensivity and precision for aptamers selection. Then it could be concluded that ELASA method could be used as a versatile, inexpensive procedure for in vito evaluation of isolated aptamers from cell-SELEX based process.

Aptamer as capture agent in enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of Aflatoxin B1.

Aflatoxin B1 (AFB1), is one of the most toxic mycotoxins found to contaminate various food commodities like cereals, dried fruits, tree nuts, spices and crude vegetable oils. In spite of considerable progress in analytical techniques, there is still a need to develop rapid and highly sensitive detection platforms for AFB1. In this study, AFB1 specific aptamer was used as a capture molecule to develop an enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of AFB1. Under optimized conditions, the assay had a linear detection range from 1 µg to 1 pg with a limit of detection (LOD) of 1 pg/mL in buffer. Conventional ELISA with AFB1 hapten as the capture agent (LOD = 10 pg/mL) was also carried out to compare the results with the present method. Recovery studies in food samples like dried red chillies, groundnut and pepper using both the methods was found to be in the range of 88.49-106.4% at 10 ng/mL and 87.4% to 95.8% at 5 ng/mL for ELASA and 76.56-127.68% at 10 ng/mL and 82-101.2% at 5 ng/mL for ELISA. Higher detection (10 fold) and better recovery using ELASA suggest that the method could offer an early, ultrasensitive, high-throughput, qualitative and semi-quantitative detection of AFB1 in contaminated food samples.

Aptamer-nanobody based ELASA for specific detection of Acinetobacter baumannii isolates.

Acinetobacter baumannii has turned into an important threat in nosocomial outbreak infections and multidrug resistance leading to high mortality rates in the 21st century. In recent years its mortality has increased by 15% which in part could be due to lack of a rapid and sensitive diagnostic test. In this work we introduced a new detection test for A. baumannii with two highly specific aptamer and nanobody molecules. High binding affinity DNA oligonucleotide aptamers toward A. baumannii were selected through 12 rounds of whole cell System Evolution of Ligands by EXponential enrichment process (SELEX). The SELEX procedures was monitored by flow cytometry. The dissociation constant and binding efficiency of the selected aptamer Aci49 was 7.547±1:353pM and 47.50%, respectively. A sandwich enzyme linked aptamer sorbent assay (ELASA) was designed with the biotinylated Aci49 aptamer and our previously developed nanobody against biofilm associated protein (Bap). The assay system was optimized with A. baumannii (ATCC 19606) and 47 clinical isolates of A. baumannii were tested. The threshold of detection in sandwich ELASA process was10(3) CFU/ml. The sensitivity of test toward the clinical isolates was 95.47%. Our results reveal that the sandwich ELASA is sensitive and specific enough for the rapid detection of A. baumannii from clinical isolates.