Selection of a new aptamer targeting amoxicillin for utilization in a label-free electrochemical biosensor.

Pharmaceutical pollution has received considerable attention because of the harmful effects of pharmaceutical compounds on human health, even in trace amounts. Amoxicillin is one of the frequently used antibiotics that was included in the list of emerging water pollutants. Therefore, a highly selective and rapid technique for amoxicillin detection is required. In this work, a new aptamer was selected for amoxicillin and utilized for the development of a label-free electrochemical aptasensor. Aptamer selection was performed using the systematic evolution of ligands by exponential enrichment. The selected aptamer showed good specificity against other antibiotics, including the structurally related antibiotics: ampicillin and ciprofloxacin. Among the selected aptamers, Amx3 exhibited the lowest dissociation constant value of 112.9 nM. An aptasensor was developed by immobilization of thiolated Amx3 aptamer onto gold screen-printed electrodes via self-assembly, which was characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The detection was realized by monitoring the change in the differential pulse voltammetry peak current in the ferro/ferricyanide redox couple upon binding of the aptasensor to amoxicillin. The aptasensor showed very good sensitivity with an ultralow limit of detection of 0.097 nM. When the aptasensor was tested using actual spiked milk samples, excellent recovery percentages were observed. The label-free electrochemical aptasensor developed herein is a promising tool for the selective and sensitive detection of amoxicillin in environmental samples.

Exploring various carbon nanomaterials-based electrodes modified with polymelamine for the reagentless electrochemical immunosensing of Claudin18.2.

Claudin18.2 (CLDN18.2) is a tight junction protein often overexpressed in various solid tumors, including gastrointestinal and esophageal cancers, serving as a promising target and potential biomarker for tumor diagnosis, treatment assessment, and prognosis. Despite its significance, no biosensor has been reported to date for the detection of CLDN18.2. Here, we present the inaugural immunosensor for CLDN18.2. In this study, an amine-rich conducting polymer of polymelamine (PM) was electrografted onto different carbon nanomaterial-based screen-printed electrodes (SPEs), including carbon (C), graphene (Gr), graphene oxide (GO), carbon nanotube (CNT), and carbon nanofiber (CNF) via cyclic voltammetry. A comparative study was performed to explore the best material for the preparation of the PM-modified electrodes to be used as in-situ redox substrate for the immunosensor fabrication. The surface chemistry and structural features of pristine and PM-deposited electrodes were analyzed using Raman and scanning electron microscopy (SEM) techniques. Our results showed that the PM deposited on Gr and CNT/SPEs exhibited the most significant and stable redox behavior in PBS buffer. The terminal amine moieties on the PM-modified electrode surfaces were utilized for immobilizing anti-CLDN18.2 monoclonal antibodies via N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide chemistry to construct the electrochemical immunosensor platform. Differential pulse voltammetry-based immunosensing of CLDN18.2 protein on BSA/anti-CLDN18.2/PM-Gr/SPE and BSA/anti-CLDN18.2/PM-CNT/SPE exhibited excellent selectivity against other proteins such as CD1, PDCD1, and ErBb2. The limits of detection of these two immunosensor platforms were calculated to be 7.9 pg/mL and 0.104 ng/mL for the CNT and Gr immunosensors, respectively. This study demonstrated that the PM-modified Gr and CNT electrodes offer promising platforms not only for the reagentless signaling but also for covalent immobilization of biomolecules. Moreover, these platforms offer excellent sensitivity and selectivity for the detection of CLDN18.2 due to its enhanced stable redox activity. The immunosensor demonstrated promising results for the sensitive detection of CLDN18.2 in biological samples, addressing the critical need for early gastric cancer diagnosis.

Aptamer-based biosensors for the detection of neonicotinoid insecticides in environmental samples: A systematic review.

Neonicotinoids, sometimes abbreviated as neonics, represent a class of neuro-active insecticides with chemical similarities to nicotine. Neonicotinoids are the most widely adopted group of insecticides globally since their discovery in the late 1980s. Their physiochemical properties surpass those of previously established insecticides, contributing to their popularity in various sectors such as agriculture and wood treatment. The environmental impact of neonicotinoids, often overlooked, underscores the urgency to develop tools for their detection and understanding of their behavior. Conventional methods for pesticide detection have limitations. Chromatographic techniques are sensitive but expensive, generate waste, and require complex sample preparation. Bioassays lack specificity and accuracy, making them suitable as preliminary tests in conjunction with instrumental methods. Aptamer-based biosensor is recognized as an advantageous tool for neonicotinoids detection due to its rapid response, user-friendly nature, cost-effectiveness, and suitability for on-site detection. This comprehensive review represents the inaugural in-depth analysis of advancements in aptamer-based biosensors targeting neonicotinoids such as imidacloprid, thiamethoxam, clothianidin, acetamiprid, thiacloprid, nitenpyram, and dinotefuran. Additionally, the review offers valuable insights into the critical challenges requiring prompt attention for the successful transition from research to practical field applications.

Redox probe-free electrochemical immunosensor utilizing electropolymerized melamine on reduced graphene oxide for the point-of-care diagnosis of gastric cancer.

Pepsinogen I (PG I) is a biomarker that plays a crucial role in the diagnosis of gastric cancer. The development of biosensor to monitor PG I overexpression in serum is crucial for early gastric cancer diagnosis, offering a less invasive alternative to the costly and uncomfortable gastroscopy procedure. This study presents a cost-efficient, scalable and disposable label-free biosensing strategy for detecting PG I, utilizing a redox-active polymelamine electrodeposited on a reduced graphene oxide screen-printed electrode surface (PM-rGO/SPE). Under optimized conditions, the conducting polymer PM was deposited on the rGO/SPE via a potentiodynamic method. The structural and morphological features of PM-rGO/SPE were analyzed with the assistance of Raman and Scanning Electron Microscopy analysis. Specific monoclonal anti-PG I antibodies were immobilized on the in situ prepared redox-active layer via EDC/NHS chemistry to develop a novel electrochemical immunosensor. Unlike the traditional immunosensing strategies which utilizes external redox probe solution for measuring the signal, the developed configuration allowed for redox-probe free monitoring of current changes of the redox active PM resulting from the formation of the immunocomplex on the electrode surface. Utilizing this method, PG I detection spanned a clinically relevant concentration range of 0.01-200 ng/mL, with a low limit of detection at 9.1 pg/mL. The electrochemical immunosensor demonstrated specificity against other biomarkers such as PDCD1, ErBb2, and CD28 with negligible interference. The immunosensor exhibited excellent recovery capabilities for PG I detection in serum samples. These findings underscore the potential of the PM-rGO/SPE immunosensor as a point-of-care diagnostic tool for gastric cancer.

Paper-Based Biosensor for the Detection of Sepsis Using MMP-9 Biomarker in FIP Mice Model.

Sepsis is an immune response to a microbial invasion that causes organ injury and dysfunction due to a systemic inflammatory response. Sepsis is a serious, life-threatening condition and a widely recognized global health challenge. Given its high death rate, it is critical to diagnose sepsis and start treatment as early as possible. There is an urgent need for a sensitive and rapid screening method for detecting sepsis. In this study, we investigated the use of MMP-9 as a biomarker for sepsis. A colorimetric paper-based biosensor was used for the detection of MMP-9 utilizing peptide-magnetic nanoparticle conjugates. The method is based on the cleavage of the MMP-9-specific peptide by the protease leading to the detaching of the magnetic beads from the sensor surface and changing of color. A fecal intraperitoneal (FIP) challenge was used to induce sepsis in mice, and an MMP-9 secretion was measured by taking blood and Bronchoalveolar Lavage (BAL) fluid samples at 1 h, 2 h, 4 h, and 20 h (early sepsis) post-challenge intervals. The results of the paper-based sensor for the detection of MMP-9 levels in blood samples and BAL samples were compared with ELISA and Western Blot. We found that both blood and BAL levels of MMP-9 increased immediately and could be detected as early as 1 h in FIP mice post-challenge. Our work adds evidence to the assertion that MMP-9 is a reliable biomarker for the detection of sepsis at early stages.

Biosensing Platform for the Detection of Biomarkers for ALI/ARDS in Bronchoalveolar Lavage Fluid of LPS Mice Model.

Acute respiratory distress syndrome (ARDS) is a worldwide health concern. The pathophysiological features of ALI/ARDS include a pulmonary immunological response. The development of a rapid and low-cost biosensing platform for the detection of ARDS is urgently needed. In this study, we report the development of a paper-based multiplexed sensing platform to detect human NE, PR3 and MMP-2 proteases. Through monitoring the three proteases in infected mice after the intra-nasal administration of LPS, we showed that these proteases played an essential role in ALI/ARDS. The paper-based sensor utilized a colorimetric detection approach based on the cleavage of peptide-magnetic nanoparticle conjugates, which led to a change in the gold nanoparticle-modified paper sensor. The multiplexing of human NE, PR3 and MMP-2 proteases was tested and compared after 30 min, 2 h, 4 h and 24 h of LPS administration. The multiplexing platform of the three analytes led to relatively marked peptide cleavage occurring only after 30 min and 24 h. The results demonstrated that MMP-2, PR3 and human NE can provide a promising biosensing platform for ALI/ARDS in infected mice at different stages. MMP-2 was detected at all stages (30 min-24 h); however, the detection of human NE and PR3 can be useful for early- (30 min) and late-stage (24 h) detection of ALI/ARDS. Further studies are necessary to apply these potential diagnostic biosensing platforms to detect ARDS in patients.

Combination of Carbon Nanofiber-Based Electrochemical Biosensor and Cotton Fiber: A Device for the Detection of the Middle-East Respiratory Syndrome Coronavirus.

The miniaturization of biosensors for point-of-care diagnosis is highly important in infection control. Electrochemical biosensors offer several advantages in diagnosis in terms of cost, disposability, portability, and sensitivity. Here, a miniaturized electrochemical immunosensor combined with cotton fiber for the detection of the Middle-East respiratory syndrome coronavirus (MERS-CoV) is described. Taking advantage of the absorption capability of cotton, the nasal and saliva samples can be collected and directly transferred to the immunosensor surface for detection using a single tool. The immunosensor was fabricated on a disposable screen-printed electrode precoated with carbon nanofibers. The electrodes were functionalized with carboxyphenyl groups that were used for the immobilization of the spike protein of the MERS-CoV. A competitive detection scheme was employed using the antibody for the MERS-CoV spike protein, and the square-wave voltammetry technique was used for measurements. The biosensor tested after the cotton coating of the electrode exhibited excellent performance. The biosensor was capable of detecting the MERS-CoV spike protein within a concentration range from 0.1 pg·mL-1 to 1 µg·mL-1 with a limit of detection of 0.07 pg·mL, implying the high sensitivity of the method. The immunosensor did not exhibit any cross-reactivity against proteins from HCoV and Influenza A, indicating the excellent selectivity of this approach. Testing of the biosensor in nasal samples showed very high recovery percentages. This disposable biosensor can be used as a miniaturized device for the collection of samples and detection of the virus using a portable potentiostat connected to a smartphone.

Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen.

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.

Determination of minimal sequence for zearalenone aptamer by computational docking and application on an indirect competitive electrochemical aptasensor.

Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (Kd = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor.

Development of a Low-Cost Cotton-Tipped Electrochemical Immunosensor for the Detection of SARS-CoV-2.

Collection of nasopharyngeal samples using swabs followed by the transfer of the virus into a solution and an RNA extraction step to perform reverse transcription polymerase chain reaction (PCR) is the primary method currently used for the diagnosis of COVID-19. However, the need for several reagents and steps and the high cost of PCR hinder its worldwide implementation to contain the outbreak. Here, we report a cotton-tipped electrochemical immunosensor for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus antigen. Unlike the reported approaches, we integrated the sample collection and detection tools into a single platform by coating screen-printed electrodes with absorbing cotton padding. The immunosensor was fabricated by immobilizing the virus nucleocapsid (N) protein on carbon nanofiber-modified screen-printed electrodes which were functionalized by diazonium electrografting. The detection of the virus antigen was achieved via swabbing followed by competitive assay using a fixed amount of N protein antibody in the solution. A square wave voltammetric technique was used for the detection. The limit of detection for our electrochemical biosensor was 0.8 pg/mL for SARS-CoV-2, indicating very good sensitivity for the sensor. The biosensor did not show significant cross-reactivity with other virus antigens such as influenza A and HCoV, indicating high selectivity of the method. Moreover, the biosensor was successfully applied for the detection of the virus antigen in spiked nasal samples showing excellent recovery percentages. Thus, our electrochemical immunosensor is a promising diagnostic tool for the direct rapid detection of the COVID-19 virus that requires no sample transfer or pretreatment.

Diagnostic techniques for COVID-19 and new developments.

COVID-19 pandemic is a serious global health issue today due to the rapid human to human transmission of SARS-CoV-2, a new type of coronavirus that causes fatal pneumonia. SARS -CoV-2 has a faster rate of transmission than other coronaviruses such as SARS and MERS and until now there are no approved specific drugs or vaccines for treatment. Thus, early diagnosis is crucial to prevent the extensive spread of the disease. The reverse transcription-polymerase chain reaction (RT-PCR) is the most routinely used method until now to detect SARS-CoV-2 infections. However, several other faster and accurate assays are being developed for the diagnosis of COVID-19 aiming to control the spread of infection through the identification of patients and immediate isolation. In this review, we will discuss the various detection methods of the SARS-CoV-2 virus including the recent developments in immunological assays, amplification techniques as well as biosensors.

Probing the influence of graphene oxide sheets size on the performance of label-free electrochemical biosensors.

The integration of graphene materials into electrochemical biosensing platforms has gained significant interest in recent years. Bulk quantities of graphene can be synthesized by oxidation of graphite to graphite oxide and subsequent exfoliation to graphene oxide (GO). However, the size of the resultant GO sheets changes from the parent graphite yielding a polydispersed solution of sizes ranging from a few nanometers to tens of micrometers. Here, we investigate the direct effect of GO sheets sizes on biosensor performance. We separated different GO sheets sizes, and we characterized them via atomic force, scanning electron, Raman and X-ray photoelectron spectroscopies and solid state nuclear magnetic resonance (NMR). As proof of concept, the sensing performance of these GO samples was probed using a well-known ssDNA aptasensor against microcystin-LR toxin and an immunosensor against ß-lactoglobulin. The resulting aptasensors and immunosensors are fabricated by using covalent attachment and physical adsorption. We found that the aptasensors fabricated using physical adsorption, the binding signal variation was dramatically increased with increasing the GO sheet size. In contrast, for the aptasensor fabricated using covalent immobilization, the binding signal variation decreased with increasing GO sheet size. However, for the ß-lactoglobulin immunosensors, the optimum signals were observed at intermediate GO sheet size. GO sheet size could enhance or inhibit the sensitivity of the graphene-based electrochemical sensors. Our results demonstrate that controlling the size of GO sheets may have a profound impact in specific biosensing applications.

Ultrasensitive peptide-based multiplexed electrochemical biosensor for the simultaneous detection of Listeria monocytogenes and Staphylococcus aureus.

A novel electrochemical biosensor is reported for simultaneous detection of two of the most common food-borne pathogens: Listeria monocytogenes and Staphylococcus aureus. The biosensor is composed of an array of gold nanoparticles-modified screen-printed carbon electrodes on which magnetic nanoparticles coupled to specific peptides were immobilized via streptavidin-biotin interaction. Taking advantage of the proteolytic activities of the protease enzymes produced from the two bacteria on the specific peptides, the detection was achieved in 1 min. The detection was realized by measuring the percentage increase of the square wave voltammetric peak current at 0.1 V versus a Ag/AgCl reference electrode in ferro/ferricyanide redox couple after incubation with the bacteria protease. The integration of the specificity of the bacterial enzymes towards their peptide substrates with the sensitivity of the electrochemical detection on the sensor array allows the rapid, sensitive and selective quantification of the two bacteria. Outstanding sensitivities were achieved using this biosensor array platform with limit of detection of 9 CFU mL-1 for Listeria monocytogenes and 3 CFU mL-1 for Staphylococcus aureus. The multiplexing capability and selectivity of the array voltammetric biosensor were demonstrated by analysing samples of Staphylococcus aureus, Listeria monocytogenes or E. coli and also containing a mixture of two or three bacteria. Using this biosensor, the two bacteria were successfully quantified simultaneously in one step without the need for DNA extraction or amplification techniques. This platform offers promise for rapid, simple and cost-effective simultaneous detection of various bacteria. Graphical abstract.

Selection, characterization, and electrochemical biosensing application of DNA aptamers for sepiapterin.

Sepiapterin reductase deficiency (SR) is a rare inborn disorder of neurotransmitter metabolism. The early diagnosis of SR disease should be achieved through the determination of the sepiapterin level in body fluids of suspected patients. Here, we report the selection, identification, and characterization of DNA aptamers against sepiapterin. The aptamer selection was achieved via the systematic evolution of ligand by the exponential enrichment technique. After ten rounds of selection, high-affinity aptamers were identified. The binding affinities of the selected aptamers were evaluated using fluorescence binding assays showing dissociation constants ranging from 37.3 to 79.0 nM. The highest affinity aptamer was then integrated into a competitive electrochemical biosensor. The biosensor achieved outstanding sensitivity with a detection limit of 0.8 pg/ml which was much lower than the reported chromatographic method for sepiapterin quantification. The aptasensor has also shown a high degree of selectivity against the closely-related compound. The aptasensor was then challenged by detecting the sepiapterin in spiked serum samples where a good recovery percentage was achieved.

A dual electrochemical/colorimetric magnetic nanoparticle/peptide-based platform for the detection of Staphylococcus aureus.

Point-of-care facile and economical detection of Staphylococcus aureus (S. aureus), one of the main causes of food-borne illness, is highly demanded for the early diagnosis and control of infections. Herein, inspired by the proteolytic activity of S. aureus protease on a specific peptide substrate, we developed a rapid, simple and cost-effective biosensor for S. aureus using dual colorimetric and electrochemical detection on the same platform. In this approach, gold screen printed electrodes were used on which specific peptide sequences coupled to magnetic nanoparticles were immobilized giving the black color of the sensor surface. The addition of the S. aureus protease solution on the electrode surface causes cleavage of the peptide sequence and the release of the magnetic nanoparticles revealing the golden colour of the electrode which can be easily seen by the naked eye. Furthermore, square wave voltammetric signals can be detected on the same electrode in the ferro/ferricyanide redox couple. The change in the peak current after peptide cleavage was directly proportional to the concentration of S. aureus. The detection limit of the electrochemical assay was 3 CFU ml-1 after 1 min. Moreover, the biosensor was capable of specifically distinguishing S. aureus from other food- and water-borne bacteria such as E. coli and Listeria using the dual mode colorimetric and electrochemical detection. The biosensor was also tested in spiked milk and water samples showing very good recovery percentages. Thus, we believe that this dual mode biosensing platform enables the easy and accurate determination of S. aureus and holds great promise for point-of-care diagnosis.

Electrochemical determination of zearalenone using a label-free competitive aptasensor.

An electrochemical aptasensor is described for determination of the phytohormone of zearalenone (ZEA). The gold electrode was modified with ZEA via covalent attachment using cysteamine-hydrochloride and 1,4-phenylene diisocyanate linker. A truncated ZEA aptamer with a dissociation constant of 13.4 ± 2.1 nM was used in an aptasensor. The electrochemical property was investigated using square wave voltammetry for monitoring the change in the electron transfer using the ferro/ferricyanide system as redox probe. Under optimal experimental conditions, the response was best measured at a potential of 0.20 V (vs. Ag/AgCl). The signals depended on the competitive mechanism between the immobilised ZEA and free ZEA for the aptamer binding site. The aptasensor works in the range 0.01 to 1000 ng·mL-1 ZEA concentration, with a detection limit of 0.017 ng·mL-1. High degree of cross-reactivity with the other analogues of ZEA was observed, whereas none towards other mycotoxins. The aptasensor was further applied for the determination of ZEA in the extract of maize grain and showed good recovery percentages between 87 and 110%. Graphical abstract Schematic representation of the electrochemical determination of zearalenone based on indirect competitive assay. Step a Immobilisation of ZEA on the surface of gold electrode via covalent attachment, b competition for the ZEA aptamer binding site between immobilised and free ZEA, and c current signal of the binding event based on SWV technique.

In vitro selection of DNA aptamers and their integration in a competitive voltammetric biosensor for azlocillin determination in waste water.

The uncontrolled usage of veterinary antibiotics has led to their widespread pollution in waterways and milk products. Potential impact of antibiotic residues on the environment and human health such as increased antibiotic resistance of microorganisms and triggering allergic reactions in humans have been reported. In this work, we developed a highly selective and sensitive voltammetric aptasensor for on-step, sensitive and low cost detection of azlocillin antibiotic, one of the broad spectrum ß-lactam antibiotics. The successful selection of DNA aptamers against azlocillin was accomplished using systemic evolution of ligands by exponential enrichment (SELEX) method. Fluorescence-binding assays showed dissociation constant of 55 nM for one of the selected aptamers (Az9). This aptamer was used to construct a competitive voltammetric aptasensor for azlocillin. A limit of detection of 1.2 pg/mL as well as remarkable selectivity against potential interfering agents, including amoxicillin, were achieved. This signal-off competitive sensor takes 30-50 min to complete the quantification of the target antibiotic. The sensor was challenged by detecting the target directly in complex environments such as tap and waste water where good recovery percentages were achieved.

Electrochemical selection of a DNA aptamer, and an impedimetric method for determination of the dedicator of cytokinesis 8 by self-assembly of a thiolated aptamer on a gold electrode.

The autosomal recessive-hyper immunoglobulin E syndromes (AR-HIES) are inherited inborn primary immunodeficiency disorders caused mainly by mutations in the dedicator of cytokinesis 8 (DOCK8) gene. A method is described for the selection of DNA aptamers against DOCK8 protein. The selection was performed by using a gold electrode as the solid matrix for immobilization of DOCK8. This enables voltammetric monitoring of the bound DNA after each selection cycle. After eight rounds of selection, high affinity DNA aptamers for DOCK8 were identified with dissociation constants (Kds) ranging from 3.3 to 66 nM. The aptamer which a Kd of 8.8 nM was used in an aptasensor. A gold electrode was modified by self-assembly of the thiolated aptamer, and the response to the DOCK8 protein was detected by monitoring the change in the electron transfer resistance using the ferro/ferricyanide system as a redox probe. The aptasensor works in the 100 pg.mL-1 to 100 ng.mL-1 DOCK8 concentration range, has a detection limit of 81 pg.mL-1 and good selectivity over other proteins in the serum. Graphical abstractSchematic representation of an electrochemical screening protocol for the selection of DNA aptamer against dedicator of cytokinesis 8 protein using electrode as solid support for target immobilization.

Disposable electrochemical immunosensor array for the multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoylecgonine.

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL-1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.