Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene Microsphere Reporter System.

CRISPR-based diagnostics use the CRISPR-Cas system trans-cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans-cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans-cleavage activity. Further work revealed that the trans-cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 µM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.

Electrochemical Nanoaptasensor Based on Graphitic Carbon Nitride/Zirconium Dioxide/Multiwalled Carbon Nanotubes for Matrix Metalloproteinase-9 in Human Serum and Saliva.

In this study, a nanocomposite was synthesized by incorporating graphitic carbon nanosheets, carboxyl-functionalized multiwalled carbon nanotubes, and zirconium oxide nanoparticles. The resulting nanocomposite was utilized for the modification of a glassy carbon electrode. Subsequently, matrix metalloproteinase aptamer (AptMMP-9) was immobilized onto the electrode surface through the application of ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS) chemistry. Morphological characterization of the nanomaterials and the nanocomposite was performed using field-emission scanning electron microscopy (FESEM). The nanocomposite substantially increased the electroactive surface area by 205%, facilitating enhanced immobilization of AptMMP-9. The efficacy of the biosensor was evaluated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under optimal conditions, the fabricated sensor demonstrated a broad range of detection from 50 to 1250 pg/mL with an impressive lower limit of detection of 10.51 pg/mL. In addition, the aptasensor exhibited remarkable sensitivity, stability, excellent selectivity, reproducibility, and real-world applicability when tested with human serum and saliva samples. In summary, our developed aptasensor exhibits significant potential as an advanced biosensing tool for the point-of-care quantification of MMP-9, promising advancements in biomarker detection for practical applications.

A novel and ultrasensitive electrochemical immunosensor based on nanocellulose-Ti3C2Tx@ZrO2 nano framework for the detection of ovalbumin.

A versatile and highly sensitive sensing platform based on nanocellulose/MXene/ZrO2 nano framework has been developed at the surface of a glassy carbon electrode (GCE) for detecting ovalbumin (Ova). To create this innovative nano framework, dialdehyde groups were introduced onto the surface of cellulose nanofibers (CNFs), which were then decorated with MXene nanosheets and nanostructured zirconia. Nanocellulose/MXene/ZrO2 nano framework was used as electrochemical mediator and immobilization environment that provided the large surface area and 197 % increment in the electrochemical signal which allowed the Ova detection in the femtomolar range. Ovalbumin antibody was immobilized on the surface of dialdehyde cellulose nanofiber through covalent bonding between amino groups of Ova and dialdehyde groups of CNFs. The assembly process of nano framework, anti-Ova, and Ova antigen were characterized using electrochemical approaches (CV and DPV). The fabricated immunosensor is further applied to DPV detection of Ova and it demonstrated a linear response to Ova antigen in the linear range of 0.01-1000 pg/mL. With optimal experimental conditions, the detection limit, quantification limit and sensitivity of Ova were found to be 1.1 fg/mL, 0.01 pg/mL and 0.1497 µA pg/mL cm-2, respectively. The fabricated immunosensor exhibited high selectivity, reproducibility, and interference resistance and achieved excellent recoveries in real food samples spiked with Ova, indicating its potential applicability in food safety monitoring.

CeO2 nanozyme mediated RPA/CRISPR-Cas12a dual-mode biosensor for detection of invA gene in Salmonella.

This study reports a novel biosensing system that leverages recombinase polymerase amplification (RPA) in conjunction with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, integrated with a nanozyme (NZ) based on cerium dioxide (CeO2). With the integration of CeO2 NZ, a dual-mode detection platform could be developed for Salmonella detection using fluorometric and colourimetric assays. The CRISPR/Cas12a system, when activated in the presence of target DNA, could cleave the FAM-labelled probe to lead to a fluorometric response. Also, when the CeO2 NZ was introduced in the presence of H2O2, a colourimetric response was generated, directly proportional to the concentration of target DNA present. We hypothesise that adding highly reactive H2O2 within the post-CRISPR/Cas12a reaction system allows for increased release of hydroxyl free radicals within the mixture. Thus, the double recognition through NZ and the CRISPR/Cas12a system provided enhanced selectivity and sensitivity to the method. The proposed biosensor could successfully detect Salmonella at concentrations as low as 0.88 pg/µL and 1.28 pg/µL for fluorometric and colourimetric responses, respectively. Furthermore, the developed biosensor could be applied in real sample analysis of raw food samples (chicken, egg, and beef) to give a good recovery in the spiked food samples with varying concentrations of cultured bacterial DNA.

A CRISPR/Cas12a-based fluorescence aptasensor for the rapid and sensitive detection of ampicillin.

This study introduces CRISPR/Cas-based aptasensor for the highly sensitive and specific detection of the antibiotic, ampicillin. Ampicillin (AMPI) is a commonly used antibiotic for treating pathogenic bacteria and is additionally added to livestock feed in agriculture. This study can enable early detection of antibiotic residues, prevent their accumulation in the environment, and ensure compliance with food safety regulations. Herein, the aptasensor was developed with the CRISPR/Cas system by utilizing three different ampicillin-specific aptamers, each conjugated with a biotin at the 5'-end. The ssDNA activator was bound to the aptamers through complementary base pairings. The attraction of the aptamers to the ampicillin target released the bound ssDNA, causing the activation of the CRISPR/Cas system. The DNA reporter probe, labelled with Cy3 and a quencher, turns on the fluorescence signal when cleaved by the activated Cas12a through trans-cleavage measured using a fluorescence spectrophotometer at 590 nm. The fluorescence signal was linearly proportional to the ampicillin target concentration with a 0.01 nM limit of detection and a read-out time of 30 min. This aptasensor showed high sensitivity towards ampicillin even in the presence of other antibiotics. The method was also successfully implemented for ampicillin detection in spiked food samples.

Nanozymes towards Personalized Diagnostics: A Recent Progress in Biosensing.

This review highlights the recent advancements in the field of nanozymes and their applications in the development of point-of-care biosensors. The use of nanozymes as enzyme-mimicking components in biosensing systems has led to improved performance and miniaturization of these sensors. The unique properties of nanozymes, such as high stability, robustness, and surface tunability, make them an attractive alternative to traditional enzymes in biosensing applications. Researchers have explored a wide range of nanomaterials, including metals, metal oxides, and metal-organic frameworks, for the development of nanozyme-based biosensors. Different sensing strategies, such as colorimetric, fluorescent, electrochemical and SERS, have been implemented using nanozymes as signal-producing components. Despite the numerous advantages, there are also challenges associated with nanozyme-based biosensors, including stability and specificity, which need to be addressed for their wider applications. The future of nanozyme-based biosensors looks promising, with the potential to bring a paradigm shift in biomolecular sensing. The development of highly specific, multi-enzyme mimicking nanozymes could lead to the creation of highly sensitive and low-biofouling biosensors. Integration of nanozymes into point-of-care diagnostics promises to revolutionize healthcare by improving patient outcomes and reducing costs while enhancing the accuracy and sensitivity of diagnostic tools.

An electrochemical aptasensor based on AuNRs/AuNWs for sensitive detection of apolipoprotein A-1 (ApoA1) from human serum.

For early detection and diagnosis of cancer, it is essential to develop an electrochemical biosensor that is quick, accurate, and sensitive. Here, we use gold nanorod (AuNR) and gold nanowire (AuNW) nanocomposites (AuNR/AuNW/CS) as electrode modifiers on a glassy carbon electrode (GCE) to construct a sensitive label-free electrochemical aptasensor to detect ApoA1. The thiolated ApoA1-specific aptamers were immobilized onto the modified electrode surface through self-assembled monolayers. Electrochemical techniques, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were used to analyze the fabrication steps. The concentration of ApoA1 was measured with DPV on the aptasensor, with a linear range of 0.1 to 1000 pg mL-1 and a detection limit of 0.04 pg mL-1. When compared to results from ELISA tests (which have a detection limit of 80 pg mL-1), the results achieved here were over 2000 times better. The aptasensor's performance was successfully evaluated using human serum spiked with ApoA1, suggesting that it has great potential for practical application. The electrochemical apatsensor additionally demonstrated outstanding selectivity responses and strong stability toward the target analyte.

Nanozyme-based sensors for detection of food biomarkers: a review.

Nanozymes have piqued the curiosity of scientists in recent years because of their ability to demonstrate enzyme-like activity combined with advantages such as high stability, inexpensive availability, robust activity, and tunable properties. These attributes have allowed the successful application of nanozymes in sensing to detect various chemical and biological target analytes, overcoming the shortcomings of conventional detection techniques. In this review, we discuss recent developments of nanozyme-based sensors to detect biomarkers associated with food quality and safety. First, we present a brief introduction to this topic, followed by discussing the different types of sensors used in food biomarker detection. We then highlight recent studies on nanozyme-based sensors to detect food markers such as toxins, pathogens, antibiotics, growth hormones, metal ions, additives, small molecules, and drug residues. In the subsequent section, we discuss the challenges and possible solutions towards the development of nanozyme-based sensors for application in the food industry. Finally, we conclude the review by discussing future perspectives of this field towards successful detection and monitoring of food analytes.

Recent trends and developments of PCR-based methods for the detection of food-borne Salmonella bacteria and Norovirus.

In recent years, rapid detection methods such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) have been continuously developed to improve the detection of food-borne pathogens in food samples. The recent developments of PCR and qPCR in the detection and identification of these food-borne pathogens are described and elaborated throughout this review. Specifically, further developments and improvements of qPCR are discussed in detecting Salmonella and norovirus. Promising advances in these molecular detection methods have been widely used to prevent human food-borne illnesses and death caused by the food-borne pathogens. In addition, this review presents the limitations and challenges of the detection methods which include conventional culture method and conventional PCR method in detecting Salmonella and norovirus. Furthermore, several advances of qPCR such as viability PCR (vPCR) and digital PCR (dPCR) have been discussed in the detection of Salmonella and norovirus. Good practice of analysis of the food-borne pathogens and other contaminants in the food industry as well as the advancement of molecular detection methods will help improve and ensure food safety and food quality.

Nanomaterials as signal amplification elements in aptamer-based electrochemiluminescent biosensors.

Biosensor technology is experiencing an unprecedented momentum that is driven by rapid advances in material science for utilization in various fields, and is constantly evolving to develop fast, simple and sensitive sensors. Here, we present a comprehensive and concise review on electrochemiluminescent (ECL) nanomaterial-based aptasensors including challenges and applications. ECL aptasensor combines the merits of a biosensor, selectivity of an aptamer, and sensitivity of ECL to construct sensitive and selective sensors. Signal amplification processes through the incorporation of nanomaterials are essential to further enhance the sensitivity of ECL sensing systems. Nanomaterials are commonly utilized in sensor fabrication as (i) electrode modifiers, (ii) nanocarriers, (iii) nanocatalysts, and (iv) luminophores. The review highlights the effects of integrating nanomaterials such as gold nanoparticles, metal nanoparticles, metal oxides, carbon nanomaterials, metal organic frameworks, and quantum dots using various research strategies that address signal amplification to enhance the ECL signal. Furthermore, various concepts have been demonstrated with relevant figures and explanations to serve as a general overview for the design of ECL aptasensors. Lastly, we discuss the underlying problems and possibilities for ECL aptasensor research and development.

Novel nanocomposite of spiky-shaped gold nanourchins/ titanium dioxide/nafion for amplified signal and efficient electrochemiluminescence detection of ovomucoid.

This work reports on the first electrochemiluminescence (ECL) immunosensor employing a novel nanostructured composite of titanium dioxide (TiO2) and gold nanourchins (AuNU) to detect protein allergen Ovomucoid (Ovm) found in eggs. TiO2 and AuNU were dispersed in Nafion and drop-casted onto SPGE with Tris(2,2'-bipyridyl)-ruthenium (II) ([Ru(bpy)3]2+) and tri-n-propylamine (TPrA) served as strong luminophore/co-reactant pairs as a source of ECL signals. The linear range, limit of detection, reproducibility and practical applications of the sensor were assessed. The fabricated ECL immuosensor produced a promising limit of detection of as low as 0.01 pg/mL. Two linear ranges of 0.01-50 pg/mL and 100-750 pg/mL with corresponding correlation coefficients of R2 = 0.99136 and R2 = 0.97829) respectively, were determined. Despite its simple fabrication method, this label-free immunosensor also showcased excellent selectivity, reproducibility, interference-resistance and yielded outstanding recoveries between 97.42 and 104.05% of Ovm analysis in spiked real food samples.

A solid-state electrochemiluminescence aptasensor for ß-lactoglobulin using Ru-AuNP/GNP/Naf nanocomposite-modified printed sensor.

An electrochemiluminescence (ECL) aptasensor for the detection of the milk protein allergen ß-lactoglobulin (ß-LG) using nanocomposite as luminophore was fabricated. The Ru-AuNPs/GNP/Naf complex was formed by combining the Rubpy32+-AuNPs complex (Ru-AuNPs), prepared by modifying the negatively charged surface of gold nanoparticles (AuNPs) with positively charged Rubpy32+ through electrostatic interactions and the graphene nanoplatelets-Nafion (GNP/Naf) at a ratio of 2:1. The nanocomposite was coated on the surface of the screen-printed electrode (SPCE) through the film-forming properties of Nafion. A layer of chitosan (CS) was coated onto this modified electrode, and later amine-terminated ß-LG aptamers were covalently attached to the CS/Ru-AuNP/GNP/Naf via glutaraldehyde (GLUT) cross-linking. When ß-LG was incubated with the aptasensor, a subsequent decrease in ECL intensity was recorded. Under the optimal conditions, the ECL intensity of the aptasensor changed linearly with the logarithmic concentration of ß-LG, in the range 0.1 to 1000 pg/ml, and the detection limit was 0.02 pg/mL (3σ/m). The constructed aptasensor displayed simple and fast determination of ß-LG with excellent reproducibility, stability, and high specificity. Additionally, the proposed ECL aptasensor displayed high recoveries (92.5-112%) and low coefficients of variation (1.6-7.8%), when ß-LG fortified samples were analyzed. Integrating Ru-AuNPs/GNP/Naf nanocomposite in the ECL aptasensor paves the way towards a cost-effective and sensitive detection of the milk allergen ß-LG.

Recent trends in nanomaterial-based signal amplification in electrochemical aptasensors.

Ultrasensitive biosensors have become a necessity in the world of scientific research, and several signal enhancement strategies have been employed to attain exceptionally low detection limits. Nanotechnology turns out to be a strong contender for signal amplification, as they can be employed as platform modifiers, catalysts, carriers or labels. Here, we have described the most recent advancements in the utilization of nanomaterials as signal amplification components in aptamer-based electrochemical biosensors. We have briefly reviewed the methods that utilized nanomaterials, namely gold and carbon, as well as nanocomposites such as: graphene, carbon nanotubes, quantum dots, and metal-organic frameworks.

A Highly Sensitive Label-free Aptasensor Based on Gold Nanourchins and Carbon Nanohorns for the Detection of Lipocalin-2 (LCN-2).

A synergistic nanocomposite film composed of gold nanourchins (AuNU), oxidised carbon nanohorns (CNH), and chitosan functioned as an electrode modifier in the fabrication of the sensitive lipocalin-2 (LCN-2) aptasensor. The AuNUs/CNH/CS composite increased the surface area and thereby amplified the signal transduction. The amine-terminated LCN-2 aptamer was immobilised through the amide bond formed between the carboxyl group of polyglutamic acid (PGA) and the amine group of aptamer. Interaction of LCN-2 with the aptamer caused conformational changes in the structure of the aptamer. This generated higher conductivity, resulting in increased DPV peak current. The DPV signal increased with increasing concentration of LCN-2, and the change in signal was used for quantitative detection. The proposed aptasensor was able to detect LCN-2 in the linear range of 0.1 - 100.0 pg mL-1, with a low detection limit of 10 fg mL-1. The aptasensor showed high sensitivity, selectivity, reproducibility, and was able to detect LCN-2 in serum samples.

Electrochemiluminescence immunosensor for tropomyosin using carbon nanohorns/Nafion/Fe3O4@Pd screen-printed electrodes.

This study focuses on developing a highly sensitive immunosensor by immobilizing oxidized carbon nanohorns/Nafion/Fe3O4@Pd nanocomposite on carbon screen-printed electrodes (SPEs) for the detection of tropomyosin (Tro-Ag). The performance of the fabricated immunosensor was investigated via electrochemiluminescence (ECL) method, resulting from the chemical reaction between tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) and tripropylamine (TPrA), and the peak intensity is recorded at 1.0 V. The nanocomposite is able to enhance the ECL intensity of [Ru(bpy)3]2 +/TPrA system and achieves high sensitivity of 28.16 fg/mL with a dynamic working range of 28.16 fg/mL to 100 ng/ml.. Furthermore, the immunosensor demonstrated a decent stability and good repeatability for Tro-Ag detection in food products. Graphical abstract The schematic representation of the modified carbon SPE with CNHs-OH/Nafion/Fe3O4@Pd and the signal produced in the (a) absence and (b) presence of Tro-Ag.

Gold-microrods/Pd-nanoparticles/polyaniline-nanocomposite-interface as a peroxidase-mimic for sensitive detection of tropomyosin.

In this study, Gold-microrods (AuMRs), Pd-nanoparticles (PdNPs), and Polyaniline (PANI) nanocomposite-interface was fabricated on the screen-printed carbon-microelectrode (SPE). Each layer of the interface was characterised using field emission-scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV). The fabricated SPE/AuMRs/PdNPs/PANI interface demonstrated the highest electronic current and showed excellent peroxidase-mimic towards H2O2 using chronoamperometry (CA). Furthermore, the SPE/AuMRs/PdNPs/PANI interface was utilised for the construction of a highly sensitive label-free electrochemical biosensor for the detection of Tpm in seafood samples. Label-free electrochemical detection of the Tpm was performed using both CA and differential pulse voltammetry (DPV) techniques. Preliminary data showed that both methods could detect Tpm as low as 0.01 pg/mL. Moreover, the developed biosensor for the detection of Tpm demonstrated excellent selectivity, high reproducibility and longer stability with an evident potential to detect Tpm in real seafood samples.

A Label-free Cardiac Troponin T Electrochemiluminescence Immunosensor Enhanced by Graphene Nanoplatelets.

In this study, a direct and label-free immunosensor was designed and constructed by modifying the screen-printed electrode with graphene nanoplatelets (GNPs) for the detection of the cardiac troponin T (cTnT). Firstly, GNPs were drop-casted onto carbon working electrode. Monoclonal cTnT antibodies were then immobilized on the GNPs via physical adsorption; finally, BSA was introduced to block non-specific binding sites. The detection of cTnT was performed using an electrochemiluminescence (ECL) technique with tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) used as a luminophore and TPrA (tripropylamine) as a co-reactant. The ECL intensity was demonstrated to be directly proportional to the cTnT concentration where a linear range from 100 pg mL-1 to 5 fg mL-1 of the cTnT detection was established. An extremely low limit of detection was achieved to be 0.05 fg mL-1 with an outstanding specificity. Additionally, this immunosensor showed excellent percentage recovery for real samples analyses in artificially spiked human serum.

An ultra-sensitive label-free electrochemiluminescence CKMB immunosensor using a novel nanocomposite-modified printed electrode.

This study presents a novel and ultrasensitive electrochemiluminescence approach for the quantitative assessment of creatine kinase MB (CK-MB). Both carbon, carbon nano-onions (CNOs) and metal-based nanoparticles, such as gold nanoparticles (AuNPs) and iron oxide (Fe3O4), were combined to generate a unique nanocomposite for the detection of CKMB. The immunosensor construction involved the deposition of the nanocomposite on the working electrode, followed by the incubation of an antibody and a blocking agent. Tris(2,2'-bipyridyl)-ruthenium(ii) chloride ([Ru(bpy)3]2+Cl) was used as a luminophore, where tri-n-propylamine (TPrA) was selected as the co-reactant due to its aqueous immobility and luminescence properties. The analytical performance was demonstrated by cyclic voltammetry on ECL. The characterization of each absorbed layer was performed by cyclic voltammetry (CV) and chronocoulometry (CC) techniques in both EC and ECL. For further characterization of iron oxide, gold nanoparticles and carbon nano-onions, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were performed. The proposed immunosensor showcases a wide linear range (10 ng mL-1 to 50 fg mL-1), with an extremely low limit of detection (5 fg mL-1). This CKMB immunosensor also exhibits remarkable selectivity, reproducibility, stability and resistance capability towards common interferences available in human serum. In addition, the immunosensor holds great potential to work with real serum samples for clinical diagnosis.

Recent developments in colorimetric immunoassays using nanozymes and plasmonic nanoparticles.

Enzyme-linked immunosorbent assay (ELISA) is a popular detection technique for the screening and diagnosis of diseases. The sensitivity of ELISA can be increased by the incorporation of nanoparticles. Through this article, we discuss the utilization of nanoparticles in ELISA. Nanoparticles possess an intrinsic biological peroxidase-like activity which allows it to act as an enzyme mimic for the development of an improved analysis method. Different nanoparticles (gold nanoparticles, silver nanoparticles, etc.) carry different peroxidase-mimic characteristics. Besides this, nanoparticles can also perform as a colorimetric substrate in ELISA where it gives a more prominent color change compared to the commonly used colorimetric substrate TMB. This article also focuses on the mechanisms behind this color change including aggregation, in situ nanoparticle growth, seeding, and etching.

Enzyme-free Gold-silver Core-shell Nanozyme Immunosensor for the Detection of Haptoglobin.

Nanoparticles have been widely developed and shown to have intrinsic enzymatic ability, and are used in biosensors. Compared to biological enzymes used in biosensors, which are expensive and tedious to harvest, enzyme-mimic nanoparticles or nanozymes are both more stable and sensitive. An important area in this work is the development of a simple detection principle of immunosensor based on the one-step synthesis of silver nanoparticle seeded onto a gold core. The gold-silver core-shell nanoparticle acts as a peroxidase mimic, which enables them to oxidise 3,3',5,5'-tetramethylbenzidine (TMB) with H2O2, giving a colourimetric response. Herein, the analytical performance of the nanozyme is exploited to detect haptoglobin as a model analyte in a 96-well plate and measured the colourimetric product using spectrophotometer. The sensitivity of the immunosensor was as low as 100 pg mL-1. The viability of our immunosensor was shown to have good selectivity and satisfactory recovery in real serum samples.