Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

A smart paper-based electrochemical sensor for reliable detection of iron ions in serum.

The fast-growing healthcare demand for user-friendly and affordable analytical tools is driving the efforts to develop reliable platforms for the customization of therapy based on individual health conditions. In this overall scenario, we developed a paper-based electrochemical sensor for the quantification of iron ions in serum as a cost-effective sensing tool for the correct supplement administration. In detail, the working electrode of the screen-printed device has been modified with a nanocomposite constituted of carbon black and gold nanoparticles with a drop-casting procedure. Square wave voltammetry has been adopted as an electrochemical technique. This sensor was further modified with Nafion for iron quantification in serum after sample treatment with trifluoroacetic acid. Under optimized conditions, iron ions have been detected with a LOD down to 0.05 mg/L and a linearity up to 10 mg/L in standard solution. The obtained results have been compared with reference methods namely commercial colorimetric assay and atomic absorption spectroscopy, obtaining a good correlation within the experimental errors. These results demonstrated the suitability of the developed paper-based sensor for future applications in precision medicine of iron-deficiency diseases.

An origami paper-based electrochemical biosensing platform for quality control of agri-food waste in the valorization strategy.

The increasing demand for food and the need for a sustainability vision in the agri-food sector have boosted novel approaches for food management, enhancing the valorization of wastes and by-products belonging to the food industry. Herein, we present a novel paper-based origami device to assess the amount of both glucosinolate and glucose in a food waste product belonging to Brassicaceae plants, to evaluate the quality value and the correct management of waste samples. The device has been designed as an origami paper-based platform constituted of two paper-based biosensors to work synergistically in a multiplexed detection. In detail, a monoenzymatic biosensor and a bienzymatic biosensor were configured for the detection of glucose and glucosinolates, respectively, using filter paper pads preloaded with glucose oxidase and/or myrosinase. To complete the paper-based platform, the enzyme-preloaded pads were combined with office paper-based electrodes modified with Carbon black/Prussian Blue nanoparticles for the measurement of enzymatic by-product at a low applied potential (i.e., 0 V versus Ag/AgCl). Overall, this paper-based platform measured glucose and glucosinolate (i.e., sinigrin) with a linear range up to 2.5 and 1.5 mM, and detection limits of 0.05 and 0.07 mM, respectively. The repeatability corresponded to an RSD% equal to 5% by testing 10 mM of glucose, and 10% by testing 1 mM of sinigrin. The accuracy of the developed multiplex device was evaluated by recovery studies at two different levels of sinigrin, i.e., 0.25 and 0.5 mM, obtaining recoveries values equal to (111 ± 3) % and (86 ± 1) %, respectively. The multiplex detection of both glucose and glucosinolate in Brassicaceae samples evaluates the quality values of the waste sample, ensuring the quality of the re-used food product waste by using an eco-designed analytical tool. The combination of paper-based devices for quality control of food waste with the re-use of these food products represents a sustainable approach that perfectly matches sustainable agrifood practices as well as the overall approach of the circular economy.

Electroanalytical Sensor Based on Gold-Nanoparticle-Decorated Paper for Sensitive Detection of Copper Ions in Sweat and Serum.

The growth of (bio)sensors in analytical chemistry is mainly attributable to the development of affordable, effective, portable, and user-friendly analytical tools. In the field of sensors, paper-based devices are gaining a relevant position for their outstanding features including foldability, ease of use, and instrument-free microfluidics. Herein, a multifarious use of filter paper to detect copper ions in bodily fluids is reported by exploiting this eco-friendly material to (i) synthesize AuNPs without the use of reductants and/or external stimuli, (ii) print the electrodes, (iii) load the reagents for the assay, (iv) filter the gross impurities, and (v) preconcentrate the target analyte. Copper ions were detected down to 3 ppb with a linearity up to 400 ppb in standard solutions. The applicability in biological matrices, namely, sweat and serum, was demonstrated by recovery studies and by analyzing these biofluids with the paper-based platform and the reference method (atomic absorption spectroscopy), demonstrating satisfactory accuracy of the novel eco-designed analytical tool.

A paper-based electrochemical sensor for H2O2 detection in aerosol phase: Measure of H2O2 nebulized by a reconverted ultrasonic aroma diffuser as a case of study.

The outbreak of COVID-19 is caused by high contagiousness and rapid spread of SARS-CoV-2 virus between people when an infected person is in close contact with another one. In this overall scenario, the disinfection processes have been largely improved. For instance, some countries have approved no-touch technologies by vaporizing disinfectants such as hydrogen peroxide, with the overriding goal to boost the safety of the places. In the era of sustainability, we designed an electrochemical paper-based device for the assessment of hydrogen peroxide nebulized by a cost-effective ultrasonic aroma diffuser. The paper-based sensor was fabricated by modifying via drop-casting a filter paper-based screen-printed electrode with a dispersion of carbon black-Prussian Blue nanocomposite, to assess the detection of hydrogen peroxide at -0.05 V vs Ag/AgCl. The use of paper-based modified screen-printed electrode loaded with phosphate buffer allowed for monitoring the concentration of hydrogen peroxide in aerosol, without any additional sampling instrument to capture the nebulized solution of hydrogen peroxide at a concentration up to 7% w/w. Hydrogen peroxide, a reconverted ultrasonic aroma diffuser, and the paper-based electrochemical sensor assisted by smartphone have demonstrated how different low-cost technologies are able to supply an useful and cost-effective solution for disinfection procedures.

Experimental Comparison in Sensing Breast Cancer Mutations by Signal ON and Signal OFF Paper-Based Electroanalytical Strips.

The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused toward the development of emerging technologies, such as liquid biopsy. DNA-based biosensors have been largely applied in this direction, and currently, there are two main approaches based on target/probe hybridization reported in the literature, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combination with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost associated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and development stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method.

Paper-Based Strips for the Electrochemical Detection of Single and Double Stranded DNA.

The detection of double stranded DNA (dsDNA) is often associated with the use of laboratory-bound approaches and/or with the prior generation of single stranded DNA (ssDNA), making these methods not suitable for in situ monitoring, i.e., point-of-care diagnostics. Screen-printed technology, coupled to the use of triplex forming oligonucleotides (TFO) as the recognizing probes, offers a great possibility toward the development of portable analytical tools. Moreover, the continuous demand for sustainable processes and waste lowering have highlighted the role of paper-based substrates for manufacturing easy-to-use, low-cost, and sustainable electrochemical devices. In this work, filter paper and copy paper have been utilized to produce E-DNA strips. Gold nanoparticles (AuNPs) have been exploited to immobilize the methylene blue (MB)-tagged TFO and to enhance the charge transfer kinetics at the electrode surface. Both paper-based substrates have been electrochemically characterized, and in addition, the effect of the amount of waxed layers has been evaluated. The paper-based E-DNA strips have been challenged toward the detection of three model targets, obtaining 3 and 7 nM as the detection limit, respectively, for single and double stranded sequences. The repeatability of the manufacturing (homemade) process has been evaluated with a relative standard deviation of approximately 10%. The effectiveness of the filter paper-based platform has been also evaluated in undiluted serum obtaining a similar value of the detection limit (compared to the measurements carried out in buffer solution). In addition, a synthetic PCR amplified dsDNA sequence, related to HIV, has been detected in serum samples.

Carbon Black-Modified Electrodes Screen-Printed onto Paper Towel, Waxed Paper and Parafilm M®.

Herein, we evaluated the use of paper towel, waxed paper, and Parafilm M® (Heathrow Scientific, Vernon Hills, IL, USA) as alternative substrates for screen-printed sensor manufacturing. Morphological study was performed to evaluate the adhesion of the ink on these uncommon substrates, as well as the morphology of the working electrode. The electrochemical characterization was carried out using ferricyanide/ferrocyanide as redox couple. To enhance the electrochemical properties of the developed sensors, the nanomaterial carbon black was used as nanomodifier. The modification by drop casting of the working electrode surface, using a stable dispersion of carbon black, allows to obtain a sensor with improved electrochemical behavior in terms of peak-to-peak separation, current intensity, and the resistance of charge transfer. The results achieved confirm the possibility of printing the electrode on several cost-effective paper-based materials and the improvement of the electrochemical behavior by using carbon black as sustainable nanomaterial.

Screen-printed electrode modified with carbon black and chitosan: a novel platform for acetylcholinesterase biosensor development.

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 µg L(-1), and a low detection limit equal to 0.05 µg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

Organophosphorous Pesticide Detection in Olive Oil by Using a Miniaturized, Easy-to-Use, and Cost-Effective Biosensor Combined with QuEChERS for Sample Clean-Up.

Herein, we report a portable electrochemical biosensor based on butyrylcholinesterase (BChE) immobilized on carbon black (CB)-modified screen-printed electrodes (SPEs) for the detection of organophosphorous pesticides in olive oil. The BChE/CB-SPE biosensor was developed to detect paraoxon in standard solutions as well as in olive oil samples previously treated with the QuEChERS method to extract pesticides from the whole fatty matrix. The biosensor shows a linear concentration range of between 20 and 100 ppb for paraoxon both in standard solutions (phosphate buffer 0.05 M) and in olive oil extracts, with a detection limit of 6 ppb in olive oil extract, corresponding to 10% of inhibition. The accuracy of this biosensor in olive oil samples was assessed with olive oil spiked with paraoxon, obtaining satisfactory recovery values.

Phosphate Detection through a Cost-Effective Carbon Black Nanoparticle-Modified Screen-Printed Electrode Embedded in a Continuous Flow System.

An automatable flow system for the continuous and long-term monitoring of the phosphate level has been developed using an amperometric detection method based on the use of a miniaturized sensor. This method is based on the monitoring of an electroactive complex obtained by the reaction between phosphate and molybdate that is consequently reduced at the electrode surface. The use of a screen-printed electrode modified with carbon black nanoparticles (CBNPs) leads to the quantification of the complex at low potential, because CBNPs are capable of electrocatalitically enhancing the phosphomolybdate complex reduction at +125 mV versus Ag/AgCl without fouling problems. The developed system also incorporates reagents and waste storage and is connected to a portable potentiostat for rapid detection and quantification of phosphate. Main analytical parameters, such as working potential, reagent concentration, type of cell, and flow rate, were evaluated and optimized. This system was characterized by a low detection limit (6 µM). Interference studies were carried out. Good recovery percentages comprised between 89 and 131.5% were achieved in different water sources, highlighting its suitability for field measurements.

An electrochemical immunoassay for the screening of celiac disease in saliva samples.

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.

A choline oxidase amperometric bioassay for the detection of mustard agents based on screen-printed electrodes modified with Prussian Blue nanoparticles.

In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (-50 mV vs. Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.

Disposable electrochemical sensor to evaluate the phytoremediation of the aquatic plant Lemna minor L. toward Pb(2+) and/or Cd(2+).

In this work a miniaturized and disposable electrochemical sensor was developed to evaluate the cadmium and lead ion phytoremediation potential by the floating aquatic macrophyte Lemna minor L. The sensor is based on a screen-printed electrode modified "in-situ" with bismuth film, which is more environmentally friendly than the mercury-based sensor usually adopted for lead and cadmium ion detection. The sensor was coupled with a portable potentiostat for the simultaneous measurement of cadmium and lead ions by stripping analysis. The optimized analytical system allows the simultaneous detection of both heavy metals at the ppb level (LOD equal to 0.3 and 2 ppb for lead and cadmium ions, respectively) with the advantage of using a miniaturized and cost-effective system. The sensor was then applied for the evaluation of Pb(2+) or/and Cd(2+) uptake by measuring the amount of the heavy metals both in growth medium and in plant tissues during 1 week experiments. In this way, the use of Lemna minor coupled with a portable electrochemical sensor allows the set up of a model system able both to remove the heavy metals and to measure "in-situ" the magnitude of heavy metal removal.

Development of a hydrogen peroxide sensor based on screen-printed electrodes modified with inkjet-printed Prussian blue nanoparticles.

A sensor for the simple and sensitive measurement of hydrogen peroxide has been developed which is based on screen printed electrodes (SPEs) modified with Prussian blue nanoparticles (PBNPs) deposited using piezoelectric inkjet printing. PBNP-modified SPEs were characterized using physical and electrochemical techniques to optimize the PBNP layer thickness and electroanalytical conditions for optimum measurement of hydrogen peroxide. Sensor optimization resulted in a limit of detection of 2 × 10(-7) M, a linear range from 0 to 4.5 mM and a sensitivity of 762 µA ∙ mM(-1) ∙ cm(-2) which was achieved using 20 layers of printed PBNPs. Sensors also demonstrated excellent reproducibility (<5% rsd).

An ELIME assay for the rapid diagnosis of coeliac disease.

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to "blank" sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r (2) of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.

A paper-based electrochemical device for the detection of pesticides in aerosol phase inspired by nature: A flower-like origami biosensor for precision agriculture.

Pesticides are largely used at worldwide level to improve food production, fulfilling the needs of the global population which is increasing year by year. Although pesticides are beneficial for crop production, their extensive use has serious consequences for the pollution of the produced food as well as for soil and groundwaters. Indeed, it is reported that 50% of sprayed pesticides reach different destinations other than their target species, including soil, surface waters, and groundwaters. For this reason, we developed a flower-like origami paper-based device for pesticides detection in aerosol phase for precision agriculture. In detail, the paper-based electrochemical platform detects paraoxon, 2,4-dichlorophenoxyacetic acid, and glyphosate at ppb levels by measuring their inhibitory activity towards three different enzymes namely butyrylcholinesterase, alkaline phosphatase, and peroxidase enzyme, respectively. This integrated electrochemical device is composed of three office paper-based screen-printed electrodes and filter paper-based pads loaded with enzymes and enzymatic substrates. The pesticide detection is carried out by measuring through chronoamperometric technique the initial and residual enzymatic activity by using a smartphone-assisted potentiostat and evaluating the percentage of inhibition, proportional to the amount of aerosolized pesticides. This paper-based device was able to detect the three classes of pesticides in aerosol phase with limits of detection equal to 30 ppb, 10 ppb, and 2 ppb, respectively for 2,4-D, glyphosate, and paraoxon.

Nanomaterials and paper-based electrochemical devices: merging strategies for fostering sustainable detection of biomarkers.

In the last few decades, nanomaterials have made great advances in the biosensor field, thanks to their ability to enhance several key issues of biosensing analytical tools, namely, sensitivity, selectivity, robustness, and reproducibility. The recent trend of sustainability has boosted the progress of novel and eco-designed electrochemical paper-based devices to detect easily the target analyte(s) with high sensitivity in complex matrices. The huge attention given by the scientific community and industrial sectors to paper-based devices is ascribed to the numerous advantages of these cost-effective analytical tools, including the absence of external equipment for solution flow, thanks to the capillary force of paper, the fabrication of reagent-free devices, because of the loading of reagents on the paper, and the easy multistep analyses by using the origami approach. Besides these features, herein we highlight the multifarious aspects of the nanomaterials such as (i) the significant enlargement of the electroactive surface area as well as the area available for the desired chemical interactions, (ii) the capability of anchoring biorecognition elements on the electrode surface on the paper matrix, (iii) the improvement of the conductivity of the cellulose matrix, (iv) the functionality of photoelectrochemical properties within the cellulose matrix, and (v) the improvement of electrochemical capabilities of conductive inks commonly used for electrode printing on the paper support, for the development of a new generation of paper-based electrochemical biosensors applied in the biomedical field. The state of the art over the last ten years has been analyzed highlighting the various functionalities that arise from the integration of nanomaterials with paper-based electrochemical biosensors for the detection of biomarkers.

Paper-based immunoassay based on 96-well wax-printed paper plate combined with magnetic beads and colorimetric smartphone-assisted measure for reliable detection of SARS-CoV-2 in saliva.

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 µg/mL, with a detection limit equal to 0.1 µg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern.

Smartphone-assisted electrochemical sensor for reliable detection of tyrosine in serum.

Point-of-care devices have attracted a huge interest by the scientific community because of the valuable potentiality for rapid diagnosis and precision medicine through cost-effective and easy-to-use devices for on-site measurement by unskilled personnel. Herein, we reported a smartphone-assisted electrochemical device consisted of a screen-printed electrode modified with carbon black nanomaterial and a commercially available smartphone potentiostat i.e. EmStat3 Blue, for sensitive detection of tyrosine. Once optimized the conditions, tyrosine was detected in standard solutions by square wave voltammetry, achieving a linear range comprised between 30 and 500 µM, with a detection limit equal to 4.4 µM. To detect tyrosine in serum, the interference of another amino acid i.e. tryptophan was hindered using a sample treatment with an extraction cartridge. The agreement of results analyzing serum samples with HPLC reference method and with the developed smart sensing system demonstrated the suitability of this smartphone-assisted sensing tool for cost-effective and rapid analyses of tyrosine in serum samples.