Low-dimensionality carbon-based biosensors: the new era of emerging technologies in bioanalytical chemistry.

Since the last decade, carbon nanomaterials have had a notable impact on different fields such as bioimaging, drug delivery, artificial tissue engineering, and biosensors. This is due to their good compatibility toward a wide range of chemical to biological molecules, low toxicity, and tunable properties. Especially for biosensor technology, the characteristic features of each dimensionality of carbon-based materials may influence the performance and viability of their use. Surface area, porous network, hybridization, functionalization, synthesis route, the combination of dimensionalities, purity levels, and the mechanisms underlying carbon nanomaterial interactions influence their applications in bioanalytical chemistry. Efforts are being made to fully understand how nanomaterials can influence biological interactions, to develop commercially viable biosensors, and to gain knowledge on the biomolecular processes associated with carbon. Here, we present a comprehensive review highlighting the characteristic features of the dimensionality of carbon-based materials in biosensing.

An improved fabric-phase sorptive extraction protocol for the determination of seven parabens in human urine by HPLC-DAD.

An improved fabric-phase sorptive extraction (FPSE) protocol has been developed and validated herein for the simple, fast, sensitive and green determination of seven parabens-methyl paraben, ethyl paraben, propyl paraben, butyl paraben, isopropyl paraben, isobutyl paraben and benzyl paraben-in human urine samples by HPLC-DAD. The mobile phase consisted of ammonium acetate (0.05 m) and acetonitrile, while total analysis time was 13.2 min. Sol-gel poly (tetrahydrofuran) coated FPSE membrane resulted in optimum extraction sensitivity for the seven parabens. The novel FPSE medium as well as the improved and faster sample preparation procedure resulted in lower limit of detection and quantitation values in comparison with previously reported methods. The separation was carried out using an RP-HPLC method with a Spherisorb C18 column and a flow rate of 1.4 ml/min. The validation of the analytical method was carried out by means of linearity, precision, accuracy, selectivity, sensitivity and robustness. For all seven parabens, the limits of detection and quantitation were 0.003 and 0.01 µg/ml, respectively. Relative recovery rates were between 86.3 and 104%, while RSD values were <12.6 and 19.3% for within- and between-day repeatability, respectively. The method was subsequently applied to real human urine samples.

Preserving Single Cells in Space and Time for Analytical Assays.

Analytical assays performed within clinical laboratories influence roughly 70% of all medical decisions by facilitating disease detection, diagnosis, and management. Both in clinical and academic research laboratories, single-cell assays permit measurement of cell diversity and identification of rare cells, both of which are important in the understanding of disease pathogenesis. For clinically utility, the single-cell assays must be compatible with the clinical workflow steps of sample collection, sample transportation, pre-analysis processing, and single-cell assay; therefore, it is paramount to preserve cells in a state that resembles that in vivo rather than measuring signaling behaviors initiated in response to stressors such as sample collection and processing. To address these challenges, novel cell fixation (and more broadly, cell preservation) techniques incorporate programmable fixation times, reversible bond formation and cleavage, chemoselective reactions, and improved analyte recovery. These technologies will further the development of individualized, precision therapies for patients to yield improved clinical outcomes.

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes.

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS6mA) dinucleotides in the GPS6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with 6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and 6mA in another consensus sequence, GPS6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in GPSATC) and Dam methyltransferase from E. coli DH10B (6mA in G6mATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6mA in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis.

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Determination of inorganic cations in biological fluids using a hybrid capillary electrophoresis device coupled with contactless conductivity detection.

We describe the assembly of a hybrid electrophoresis device that contains fused silica capillaries interconnected to a microfabricated interface in a cross format for the determination of inorganic cations in biological samples. The sample transport in the proposed hybrid device was performed under gated injection mode and the separations were monitored with a capacitively coupled contactless conductivity detector. The capillary extremities were inserted into polypropylene tubes to create solution reservoirs. Sensing electrodes were produced using stainless-steel hypodermic needles previously cut with 2.0 mm length. The running composition and injection time were optimized and the best results were found using 50 mmol/L lactic acid, 20 mmol/L histidine and 3 mmol/L 18-crown-6 ether, and an electrokinetic injection time of 15 s. The separation of six inorganic cations was achieved with baseline resolution, and efficiencies were between 9.1 × 103 and 5.4 × 104 plates/m. The proposed hybrid device was explored for determining the concentration levels of inorganic cations in urine, saliva, and tear samples, employing Li+ as an internal standard. The achieved results were in good agreement with the data reported in the literature. The reliability of the proposed method ranged from 93 to 98%, thus suggesting satisfactory accuracy for bioanalytical applications.

Hand drawing of pencil electrodes on paper platforms for contactless conductivity detection of inorganic cations in human tear samples using electrophoresis chips.

This paper describes for the first time the fabrication of pencil drawn electrodes (PDE) on paper platforms for capacitively coupled contactless conductivity detection (C(4) D) on electrophoresis microchips. PDE-C(4) D devices were attached on PMMA electrophoresis chips and used for detection of K(+) and Na(+) in human tear samples. PDE-C(4) D devices were produced on office paper and chromatographic paper platforms and their performance were thoroughly investigated using a model mixture containing K(+) , Na(+) , and Li(+) . In comparison with chromatographic paper, PDE-C(4) D fabricated on office paper has exhibited better performance due to its higher electrical conductivity. Furthermore, the detector response was similar to that recorded with electrodes prepared with copper adhesive tape. The fabrication of PDE-C(4) D on office paper has offered great advantages including extremely low cost (< $ 0.004 per unit), reduced fabrication time (< 5 min), and minimal instrumentation (pencil and paper). The proposed electrodes demonstrated excellent analytical performance with good reproducibility. For an inter-PDE comparison (n = 7), the RSD values for migration time, peak area, and separation efficiency were lower than 2.5, 10.5, and 14%, respectively. The LOD's achieved for K(+) , Na(+) , and Li(+) were 4.9, 6.8, and 9.0 µM, respectively. The clinical feasibility of the proposed approach was successfully demonstrated with the quantitative analysis of K(+) and Na(+) in tear samples. The concentration levels found for K(+) and Na(+) were, respectively, 20.8 ± 0.1 mM and 101.2 ± 0.1 mM for sample #1, and 20.4 ± 0.1 mM and 111.4 ± 0.1 mM for sample #2.

Indicator displacement assays inside live cells.

The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells.

Quantitative analysis of organophosphorus pesticides in freshwater using an optimized firefly luciferase-based coupled bioluminescent assay.

In this paper, a coupled bioluminescent assay, relying on the coupling of the enzymes acetylcholinesterase, S-acetyl-coenzyme A synthetase and firefly luciferase, for the detection and quantitation of organophosphorus pesticides, is presented. Using malathion as a model organophosphorus pesticide, the assay was optimized through statistical experimental design methodology, namely Plackett-Burman and central composite designs. The optimized method requires only 20 µL of sample. The linear range for the assay was 2.5-15 µM of malathion, with limits of detection and quantitation of 1.5 and 5.0 µM, respectively. This simple, fast and robust method allows samples to be analyzed at room temperature and without any pretreatment.

A review of the recent achievements and future trends on 3D printed microfluidic devices for bioanalytical applications.

3D printing has revolutionized the manufacturing process of microanalytical devices by enabling the automated production of customized objects. This technology promises to become a fundamental tool, accelerating investigations in critical areas of health, food, and environmental sciences. This microfabrication technology can be easily disseminated among users to produce further and provide analytical data to an interconnected network towards the Internet of Things, as 3D printers enable automated, reproducible, low-cost, and easy fabrication of microanalytical devices in a single step. New functional materials are being investigated for one-step fabrication of highly complex 3D printed parts using photocurable resins. However, they are not yet widely used to fabricate microfluidic devices. This is likely the critical step towards easy and automated fabrication of sophisticated, complex, and functional 3D-printed microchips. Accordingly, this review covers recent advances in the development of 3D-printed microfluidic devices for point-of-care (POC) or bioanalytical applications such as nucleic acid amplification assays, immunoassays, cell and biomarker analysis and organs-on-a-chip. Finally, we discuss the future implications of this technology and highlight the challenges in researching and developing appropriate materials and manufacturing techniques to enable the production of 3D-printed microfluidic analytical devices in a single step.

Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment.

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.

Touring the Tomato: A Suite of Chemistry Laboratory Experiments.

An eight-session interdisciplinary laboratory curriculum has been designed using a suite of analytical chemistry techniques to study biomaterials derived from an inexpensive source such as the tomato fruit. A logical progression of research-inspired laboratory modules serves to "tour" the macroscopic characteristics of the fruit and the submicroscopic properties of its constituent cuticular biopolymers by atomic force microscopy (AFM), UV-visible, and nuclear magnetic resonance (NMR) methods at increasingly detailed molecular levels. The modular curriculum can be tailored for specialty undergraduate courses or summer high school workshops. By applying analytical tools to investigate biopolymers, making connections between molecular and microscale structure, and linking both structural regimes to the functional properties of natural polymers, groundwork is established for further student investigations at the interface of chemistry with biology or chemical engineering.

Imunocapture Magnetic Beads Enhanced and Ultrasensitive CRISPR-Cas13a-Assisted Electrochemical Biosensor for Rapid Detection of SARS-CoV-2.

The rapid and ongoing spread of the coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emphasizes the urgent need for an easy and sensitive virus detection method. Here, we describe an immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, collectively known as CRISPR-Cas13a technology. At the core of the detection process, low-cast and immobilization-free commercial screen-printed carbon electrodes are used to measure the electrochemical signal, while streptavidin-coated immunocapture magnetic beads are used to reduce the background noise signal and enhance detection ability by separating the excessive report RNA, and a combination of isothermal amplification methods in the CRISPR-Cas13a system is used for nucleic acid detection. The results showed that the sensitivity of the biosensor increased by two orders of magnitude when the magnetic beads were used. The proposed biosensor required approximately 1 h of overall processing time and demonstrated an ultrasensitive ability to detect SARS-CoV-2, which could be as low as 1.66 aM. Furthermore, owing to the programmability of the CRISPR-Cas13a system, the biosensor can be flexibly applied to other viruses, providing a new approach for powerful clinical diagnostics.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Simultaneous determination of aflatoxins B1, B2, G1 and G2 in commercial rices using immunoaffinity column clean-up and HPLC-MS/MS.

Rice is frequently contaminated with aflatoxins, that are highly toxic fungal substances and strongly involved on hepatic cancer. In this work, different extraction and clean-up methods were evaluated for the simultaneous extraction and clean-up of aflatoxins B1, B2, G1 and G2 from rice. Favourable results were obtained by using methanol - water (80:20, v/v) extraction followed by immunoaffinity columns for clean-up, with recoveries of 86-92%, standard deviations between 5 and 11%, LOD ranged between 0.09 and 0.32 µg/kg, and LOQ between 0.31 and 1.06 µg/kg. Method validation and sample analysis were performed by using HPLC-MS/MS. Nine rice samples from different origin, varieties and specific characteristics, acquired in Spanish supermarkets were analysed. In two basmati samples from the same batch aflatoxin B1 was detected at (1.62 ± 0.08) µg/kg and (0.77 ± 0.03) µg/kg, both lower than the levels established by European Regulation for aflatoxin B1 in cereals.

Ruggedness testing of liquid chromatography-tandem mass spectrometry system components using microbiome-relevant methods and matrices.

Recently, an opportunity to perform a broad ruggedness assessment of our liquid chromatography-tandem mass spectrometry (LC-MS/MS) system presented itself during the analytical planning phase of a large-scale human fecal microbiome study. The specific aim of this project was to study the microbial-mediated metabolism of a targeted set of bile acids/salts by mixed bacterial communities cultured from the feces of 12 healthy volunteers when grown in a custom growth medium and following exposure to different clinically-relevant antibiotics. The magnitude of this study offered a rare opportunity to significantly stress procedures and LC-MS/MS system components comprised in our bile acid/salt targeted metabolomics method. With this second specific aim in mind, we modified the sample analysis plan to include a series of figure-of-merit (FoM)-based tests that are commonly used in regulated bioanalytical labs to assess LC and MS system ruggedness for a specific assay - these FoM-based testing parameters were monitored continuously over the course of sample analysis and the results are presented in this report. In total, the assessment included 1206 sequential injections (180 calibration standards, 136 blank-internal standard samples, and 890 diluted medium samples) that took place over 8-days. Completion of the 8-days of non-stop sample analysis revealed no critical hardware or software failures, and the analysis of the FoM-based tests indicated no observable degradation of system performance over the number of samples and time tested. The FoM-based test metrics presented may be used as a template to assess the ruggedness of any LC-MS/MS-based targeted metabolomics workflow.

A high-throughput LC-MS/MS method for the measurement of the bile acid/salt content in microbiome-derived sample sets.

Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode - ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period.

Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with integrated contactless conductivity detection.

This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C4D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C4D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span® 80 were pumped through flow rates fixed at 5 and 60 µL min-1, respectively. The droplets with incorporated cells were monitored in the C4D system applying a 500-kHz sinusoidal wave with 1 Vpp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321 ±â€¯9 µm and presented volume of 17.3 ±â€¯0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 CFU droplet-1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet-1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.

Poly-adenine-mediated spherical nucleic acids for strand displacement-based DNA/RNA detection.

DNA-gold nanoparticles (AuNPs) conjugate is one of the most versatile bionanomaterials for biomedical and clinical diagnosis. However, to finely tune the hybridization ability and precisely control the orientation and conformation of surface-tethered oligonucleotides on AuNPs remains a hurdle. In this work, we developed a poly adenine-mediated spherical nucleic acid (polyA-mediated SNA) strategy by assembling di-block DNA probes on gold nanoparticles (AuNPs) to spatially control interdistance and hybridization ability of oligonucleotides on AuNPs. By modulating length of poly A bound on the SNA with different degrees of constructing, we presented significant improved biosensing performance including high hybridization efficiency, and expanded dynamic range of analytes with more sensitive detection limit. Furthermore, this polyA design could facilitate the programmable detection for DNA in serum environment and simultaneous multicolor detection of three different microRNAs associated with pancreatic carcinoma. The demonstration of the link between modulation of SNA assembly strategy and biodetection capability will increase the development of high performance diagnostic tools for translational biomedicine.