Real Blood Samples Research Articles

Determination of anabolic steroids in dried blood using microsampling and gas chromatography-tandem mass spectrometry: Application to a testosterone gel administration study.

Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL-1 in 20 μL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.

Cross-linked chitosan biofunctionalized paper-based microfluidic device towards long term stabilization of blood typing antibodies

Long term stability of antibodies at room temperature is a major challenge in the commercialization of point-of-care devices for diagnostics. Since chitosan has been proven to be an excellent biofunctionalization material, the effects of four different biofunctionalization processes were studied to improve the room temperature stability of antibodies immobilized on chitosan modified paper-based microfluidic devices using blood typing antibodies as candidates. The devices used in this work have a flower-shaped design with 4 test zones at each corner. In three zones Anti-A, Anti-B, and Anti-D (Anti-Rh) antibodies are immobilized and the fouth zone represents the control (no antibodies) after biofunctionalization. The biofunctionalization of the paper devices was done with chitosan and chitosan cross-linked with sodium triphosphate pentabasic, glutaraldehyde, and sodium hydroxide. These devices were used for blood typing assays using real blood samples. A similar assay was also performed on unmodified (non-biofunctionalized) paper devices for comparison. Chitosan based biofunctionalized paper-devices showed better stability, up to 100 days as compared to 14 days on unmodified paper, at room temperature. Such biofunctionalized paper-based devices will be suitable for on-field and remote testing without any technical expertise and requirement for the cold chain.

Employing Label‐free Electrochemical Biosensor Based on 3D‐Reduced Graphene Oxide and Polyaniline Nanofibers for Ultrasensitive Detection of Breast Cancer BRCA1 Biomarker

AbstractA label‐free DNA biosensor based on three‐dimensional reduced graphene oxide (3D‐rGO) and polyaniline (PANI) nanofibers modified glassy carbon electrode (GCE) was successfully developed for supersensitive detection of breast cancer BRCA1. The results demonstrated that 3D‐rGO and PANI nanofibers had synergic effects for reducing the charge transfer resistance (Rct), meaning a huge enhancement in electrochemical activity of 3D‐rGO‐PANI/GCE. Probe DNA could be immobilized on 3D‐rGO‐PANI/GCE for special and sensitive recognition of target DNA (1.0×10−15–1.0×10−7 M) with a theoretical LOD of 3.01×10−16 M (3S/m). Furthermore, this proposed nano‐biosensor could directly detect BRCA1 in real blood samples.

Supporting dataset on the validation and verification of the analytical method for the biomonitoring of 360 toxicologically relevant pollutants in whole blood.

The dataset presented in this article supports “Micro QuEChERS-based method for the simultaneous biomonitoring in whole blood of 360 toxicologically relevant pollutants for wildlife” (Rial-Berriel et al., 2020). The supplementary data are: (1) Detailed validation data of the LC-MS/MS and GC-MS/MS methods for the quantification of 360 chemicals covering bias and precision (intra- and inter-day variability) for retention times, linearity, and limits of quantification. (2) Graphical data of the matrix effects on the quantification of all of the analytes. (3) Individual data of the 51 chemicals detected in real whole blood samples from two raptor species: 36 barn owls (Tyto alba) and 112 common kestrels (Falco tinnunculus).

Silk cocoon membrane-based immunosensing assay for red blood cell antigen typing

Accurate and fast identification of blood groups is a critical step in the blood transfusion procedure. In this study, we reported a rapid and reliable immunosensing assay for ABO and RhD blood group typing based on natural free-standing silk cocoon membranes. Natural silk cocoon membranes were punched into discs (SMDs) and were treated with heat and detergent to increase wettability. Monoclonal antibodies (mAbs) against silk proteins were successfully prepared through animal immunization. The SMDs were functionalized by affinity binding with specific silk mAbs and crosslinking antibodies. Blood grouping antibodies were coupled with functional SMDs to form the immunosensor for blood typing. Only three pipetting steps were required to perform the assay, without the need for any external analytical equipment. Results could be obtained and distinguished by the naked eye within 30 s, making this immunosensor suitable for point-of-care testing. A positive result was obtained when the SMD showed a visualization red spot after washing, no red spot was observed if there was no reaction. We compared our sensing results with those from the gold-standard tube test on 112 real blood samples. The accuracy was 100 %. Due to the availability of the cocoon membranes-based immunosensor and the facile fabrication process, the SMART can be scaled up easily for mass production.

Determination of Illicit Drugs in Dried Blood Spots by LC–MS/MS Method: Validation and Application to Real Samples

Drugs of abuse can be investigated in dried blood spot (DBS) samples to evaluate the intake of psychoactive drugs. We have developed the validated liquid chromatography–tandem mass spectrometry method to determine drug of abuse in whole blood with DBS. The mass spectrometer was performed using multiple reaction monitoring via positive electro-spray ionization. The analysis procedure was applied to real positive blood samples of the 59 cases brought to Forensic Toxicology Laboratory in Faculty of Medicine of Cukurova University by law enforcement agencies. The method based on liquid–liquid extraction and liquid chromatography tandem mass spectrometry was fully validated according to international guidelines and then applied to authentic positive blood samples. Overall, polydrug usage was determined in 24 real samples and one or more psychoactive drugs were found positive in 51 of 59 real samples, which were found to be positive via performing routine whole blood analysis and selected for DBS analysis. Our method developed could applicate as an alternative to conventional analytical and sampling techniques and respond to some of the present issues concerning the different forensic toxicological investigations.

Co(OH)2 nanosheets decorated Cu(OH)2 nanorods for highly sensitive nonenzymatic detection of glucose

Co(OH)2 nanosheets/Cu(OH)2 nanorods composite electrodes for non-enzymatic glucose detection were fabricated by electrodepositing Co(OH)2 nanosheets on Cu(OH)2 nanorods substrate grown directly on the copper sheet via a simple one-step reaction. The Co(OH)2 nanosheets/Cu(OH)2 nanorods composite electrode was characterized by scanning electron microscopy, energy dispersive x-ray spectroscopy, x-ray diffraction and x-ray photoelectron spectroscopy. The glucose sensing performance of the composite electrode was investigated by cyclic voltammetry and chronoamperometry. The composite electrode shows high sensitivity of 2366 µA mM−1 cm−2 up to 2 mM with a lower detection limit of 0.17 mM (S/N = 3). The composite electrode is highly selective to glucose in the presence of various substances that always co-exist with glucose in real blood samples. The response of the composite towards human blood serum was in good agreement with that of commercially available glucose sensors, suggesting that a promising electrode material for highly sensitive and selective non enzymatic detection of glucose can be envisioned.

Accurate prediction of somatic variants using deep learning model.

e13659 Background: Efficient and accurate identification of somatic variant is important for understanding the formation, progression, and treatment of cancer. It is necessary to conduct manual review by Integrative Genomic Viewer (IGV) in traditional variant calling process. However, the traditional manual is heavy workload when evaluating tumor with a high variant burden. In this study, a new convolutional neural network (CNN) method was created to train models for somatic mutation identification, which was suitable for Panel sequencing platform with different tumor purities. Methods: A total of 1000 tumor samples from next generation sequencing (NGS)-based genetic testing by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Through variant calling program, like GATK, the candidate mutation locations were identified and standardized by manual confirmation. For each candidate mutation location, reads of both tumor and control tissue were extracted. A 2-dimensional feature matrix M of size (2k+1) * 32 in each candidate base was created. The rows of 2k+1 represented the length of candidate region, and the 32 columns included the reads coverage frequency, mapping quality messages, and genome local scores of different tumor and control tissues. CNN model, which includes nine convolutional layers structured by Temporal Convolutional Networks (TCN) but with a different structure to adapt to the proposed input matrix, was used for training. The training data set including manually validated sequence data was used as benchmark test, and optimized by Stochastic Gradient Descent (SGD) optimizer with a learning rate of 0.01 was used for training. Results: The validation data set included 15 mixed samples which were composed of different proportions of known cell lines and real mixed blood samples. The pooled DNA contained 2,359 somatic variants, with expected variant allele frequencies ranged from 3% to 97% in each pool. The overall sensitivity and positive predictive value (PPV) of single nucleotide variants (SNVs) were 99.3% and 99.8%, respectively. Conclusions: A novel and sensitive computational tool for somatic variation detection in DNA Panel sequencing was developed. Our result showed that the deep learning CNN model could call variant in Panel sequencing data.

CRISPR-dCas9 powered impedimetric biosensor for label-free detection of circulating tumor DNAs

Label-free biosensors which can be integrated into lab-on-a-chip platforms have the advantage of using small volumes for rapid and inexpensive measurements contrary to label-based technologies which are often more costly and time-consuming. In this study, graphene oxide screen printed electrodes (GPHOXE) were modified by deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor for label-free detection of circulating tumor DNAs (ctDNA). This was achieved by detection of a tumor related mutation (PIK3CA exon 9 mutation) via sequence-specific recognition followed by electrochemical impedance spectroscopy (EIS) analysis. The biosensor showed high specificity as there was no impedance signal for other ctDNA sequences, even the single nucleotide mismatch. dCas9-sgRNA modified biosensor demonstrated linear detection limits between 2 and 20 nM for 120 bp ctDNA’s in 40 s. The calibration curve showed good linearity, LOD was calculated as 0.65 nM and LOQ was calculated as 1.92 nM. Selectivity and repeatability studies were carried out in real blood samples and the recovery was higher than 96%. In conclusion, dCas9-sgRNA was effectively immobilized and optimized on GPHOXE as the selective biorecognition receptor of this ultrafast impedimetric biosensor. The CRISPR-dCas9 powered impedimetric system showed good selectivity, high repeatability and good recovery properties. This is the first literature to report the use of CRISPR/Cas technology as a label-free tool that can be used in an impedimetric system for detection of ctDNA’s.

Determination of Selenium in Biological Samples by Flame Atomic Absorption Spectrometry after Preconcentration on Modified Polyurethane Foam

An analytical method has been proposed based on the separation and determination of selenium(IV) ion using polyurethane foam as a solid sorbent coated with ammonium pyrrolidine dithiocarbamate (APDC). Different factors including the pH of sample solution, time of extraction, volume of sample, and amount of the loaded and unloaded polyurethane foam were examined and a preconcentration factor of 5 was obtained. The interference effects of some additional salts in the solution such as NaCl, FeCl3, BaCl2, CH3COONa and Na2SO4 have been investigated. The improved limit of detection (LOD) for the method is 0.064 mg/L. The improved LOD is much lower than the LOD of FAAS and approaching the LOD of GFAAS. The new developed procedure SPE-FAAS has been Found to be successful in separating the complex matrices (human hair, bovine liver, pork liver, bovine muscle) and pre-concentrating selenium ion from real blood sample. The developed method for solid-liquid extraction is convenient, simple, sensitive and of low costs.

Hollow-fibre liquid-phase microextraction and gas chromatography-mass spectrometric determination of amphetamines in whole blood

Here, we present a fully validated method using a hollow-fibre liquid-phase microextraction technique for the determination by gas chromatography-mass spectrometry (GC–MS) of amphetamine (AMP), methamphetamine (MET), fenproporex (FEN), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine (MDEA) in whole blood. The validation parameters presented successful values within those recommended by the Scientific Working Group for Forensic Toxicology (SWGTox) in the Standard Practices for Method Validation in Forensic Toxicology. The limits of detection ranged from 1 to 3 ng/mL, and the limits of quantification ranged from 2 to 5 ng/mL. The determination coefficients (r2) ranged from 0.990 to 0.997, and the method presented good intraday and interday accuracy (from 90.4% to 97.2%) and satisfactory recovery (from 68% to 110%). No carryover was observed. The heteroscedasticity was tested, and only AMP presented homoscedasticity. Weighting factors were applied to correct the linearity of MET (1/x2), MDA (1/x), FEN (1/x1/2), MDMA (1/x2) and MDEA (1/y). Dilution integrity was tested at ratios of 1:2, 1:5 and 1:10, and all maintained intraday precision (from 94.9% to 99.3%) and interday precision (from 89.4% to 94.9%). The validated method was applied to six real whole blood samples from individuals suspected of consuming ecstasy, and MDMA, MDA and amphetamine were successfully identified and quantified.

A Novel Chronoimpedimetric Glucose Sensor in Real Blood Samples Modified by Glucose‐imprinted Pyrrole‐Aminophenylboronic Acid Modified Screen Printed Electrode

AbstractIn this study, a molecularly imprinted sensor technology is engineered to detect glucose in real blood samples by chronoimpedimetrically. The imprinting process of glucose (Glc) was carried out by electrochemical polymerization of aminophenylboronic acid (APBA) and pyrrole (Py) by performing cyclic voltammetry (CV). Afterwards, glucose molecule was removed from imprinted surface by 5 % acetic acid to reveal glucose imprinted cavities. Electrochemical Impedance Spectroscopy (EIS) was used to characterize sensor modification steps and glucose removal. Glucose monitoring process was carried out chronoimpedimetrically(CI) for the first time in real blood samples. Calibration curve was prepared between 20–800 mg/dL. The standard deviations of the 18 calibration curves R2 were calculated as 0.9866±0.0066 to assess reproducibility. Recovery was calculated by using 105 mg/dL Glc Serum Sample, which was monitored by auto analyzer and into this sample 50 mg/dL Glc added and our sensor response was 147.92±2.43 mg/dL, 98.6±1.62 % (n=5). Non‐imprinted (NIP) sensor gave no signal for the glucose concentration.

Competitive electrochemical immunosensor for the detection of unfolded p53 protein in blood as biomarker for Alzheimer’s disease

Alzheimer's disease is one of the most common causes of dementia nowadays, and its prevalence increases over time. Because of this and the difficulty of its diagnosis, accurate methods for the analysis of specific biomarkers for an early diagnosis of this disease are much needed. Recently, the levels of unfolded isoform of the multifunctional protein p53 in plasma have been proved to increase selectively in Alzheimer's Disease patients in comparison with healthy subjects, thus entering the list of biomarkers that can be used for the diagnosis of this illness.We present here the development of an electrochemical immunosensor based on nanostructured screen-printed carbon electrodes for the quantification of unfolded p53 in plasma samples. The sensor shows a suitable linear range (from 2 to 50 nM) for its application in real blood samples and a very low limit of detection (0.05 nM). The concentration of unfolded p53 has been accurately detected in plasma of elderly people in healthy conditions, subjects with mild cognitive impairment (MCI) and Alzheimer's Disease (AD) subjects, obtaining results with no significant differences to those provided by an ELISA assay. These results support the possibility of measuring unfolded p53 levels with a cheap, simple and miniaturized device with a promising future for point-of-care applications in the early diagnosis of Alzheimer's dementia.

Molecular Crowding Evolution for Enabling Discovery of Enthalpy-Driven Aptamers for Robust Biomedical Applications.

An enthalpy-driven ligand is an ideal probe for practical applications because of the formation of abundant specific bonds between the ligand and target, compared to an entropy-driven ligand with a similar Gibbs free energy change. However, there has been a lack of direct discovery strategy for identifying enthalpy-driven ligands. In this work, a molecular crowding SELEX (systematic evolution of ligands by exponential enrichment) strategy for discovering enthalpy-driven aptamers was developed to improve the affinity and selectivity of aptamers in complex samples. Three aptamer sequences were successfully evolved against a tumor biomarker protein, and all proved to be enthalpy-driven by thermodynamics analysis, establishing the feasibility of molecular crowding SELEX for effective discovery of enthalpy-driven aptamers. Further comparison of aptamers evolved from conventional SELEX in buffer and molecular crowding SELEX (SYL-H2C) revealed much higher affinity of SYL-H2C. With its improved thermodynamic properties, the enthalpy-driven SYL-H2C aptamer was able to detect circulating tumor cells in real cancer patient blood samples with excellent detection accuracy (10/10). The proposed molecular crowding screening strategy offers a promising direction for discovering robust binding probes for a great variety of biomedical applications.

Visual simultaneous detection of single nucleotide polymorphism of tumor susceptibility gene and marker alpha-fetoprotein based on double-labeled colloidal gold probe with lateral flow strip biosensor

Liver cancer is one of the most common cancers in the world. The gene CYP1A1 is closely related to the occurrence of cancer, and the tumor marker alpha-fetoprotein (AFP) is also one of the important indicators for liver cancer screening. Therefore, detection of these two substances is of great significance. In this paper, a test strip biosensor capable of simultaneously detecting cancer susceptibility gene CYP1A1 and AFP has been established. There were two detection lines in the strip biosensor, one was used for qualitative detection of susceptible gene by base complementary pairing, and the other was used for semi-quantitative detection of AFP by antigen-antibody reaction. Finally, signal amplification was achieved by horseradish peroxidase catalyzing substrate 3-amino-9-ethylcarbazole. Results showed that under the optimized conditions, the strip biosensor had a good linear relationship (R2 = 0.987) when AFP concentration was in the range of 20–250 ng/mL, and the other hundred times of proteins had no obvious interference to the detection, indicating that the method presented good selectivity. The detection of susceptibility gene CYP1A1 and AFP in real blood samples by the strip was in accordance the standard method, which showed that the method had good accuracy and could be used for qualitative judgment of SNP locus base type of susceptibility gene and semi-quantitative detection of tumor marker in real sample, so it had a good application potential.

Separation detection of hemoglobin and glycated hemoglobin fractions in blood using the electrochemical microfluidic channel with a conductive polymer composite sensor

Separation and detection of hemoglobin (Hb) and glycated hemoglobin fractions (HbA1c, HbAld1+2, HbAle, HbAld3a, HbAla+b, HbA2, and HbAld3b) was performed using an electrochemical AC field modulated separation channel (EMSC) coupled with a sensor probe. The sensor was fabricated based on immobilization of a redox mediator on the poly(2,2′:5′,5″-terthiophene-3′-p-benzoic acid, pTTBA) and N,S-doped porous carbon (NSPC) nanocomposite. The different types of catalytic redox mediators such as Nile Blue (NB), toluidine blue O (TBO), and Neutral Red (NR) were evaluated to achieve the efficient detection. Of these, the NB-based sensor showed the best analytical signal for Hb and HbA1c, thus it was characterized using various electrochemical and surface analysis methods. After that, the sensor was coupled with the EMSC to achieve the separation detection of the Hb family. The frequency and amplitude of the AC electrical field applied onto the EMSC walls were the main driving forces for the separation and sensitive detection of the analytes. Under optimized conditions, linear dynamic ranges for Hb and HbA1c among their fractions were obtained between 1.0 × 10−6 to 3.5 mM and 3.0 × 10−6 to 0.6 mM with the detection limit of 8.1 × 10−7 ± 3.0 × 10−8 and 9.2 × 10−7 ± 5 × 10−8 mM, respectively. Interference effects of other biomolecules were also investigated and the clinical applicability of the device was evaluated by the determination of total Hb and % HbA1c in real human blood samples.

Electrochemical Glucose Sensor based on Microporous Carbon/CuO@Carbon/AuNPs Integrated Electrode

AbstractA novel nonenzymatic glucose sensor based on CuO@Carbon nanostructures capped with gold nanoparticles (AuNPs) arrayed on three‐dimensional porous carbon derived from kenaf stem (3D‐KSC) was proposed. Here, the massive 3D‐KSC with honeycomb‐like structure was used as supporting matrix to load CuO@Carbon nanostructures. A small amount of AuNPs were coated on CuO@Carbon nanostructures, which were arrayed on 3D‐KSC uniformly. The AuNPs improved the electrical conductivity and catalytic activity of CuO@Carbon nanostructures greatly. Thus, the 3D‐KSC/CuO@Carbon/AuNPs integrated electrode based nonenzymatic glucose sensor showed good performance with the linear range of 3.71 μM–8.5 mM, the detection limit of 1.22 μM and the sensitivity of 935 μA cm−2 mM−1. The satisfied performance made sure it can be used to detect glucose in real blood samples.

A novel computational tool for copy number variation detection in targeted circulating tumor DNA.

e13051 Background: Targeted sequencing of circulating tumor DNA (ctDNA) has been used in early tumor detection and guidance of clinical cancer treatment. Accurate somatic copy number variation (SCNV) estimation is useful for better decisions in clinics, but remain challenging due to low percentage of tumor-cell-released DNA in circulating blood, in addition to the low signal-noise ratio and usual lack of normal control. Methods: To overcome these challenges and call SCNV at gene level, we develop a novel bioinformatics tool ctCNV. After correction for GC content, target region length and read counts, genome local scores at the gene level (GCS) were calculated, using 30 normal blood samples as control. Statistically significant cutoffs were determined using control. Results: The new method has been benchmarked with other SCNV calling tools such as CNVkit, which have been developed mainly for whole exome sequencing instead of targeted DNAseq. The validation experiment involves 6 mixed samples consisting of different proportions of known cell lines and real blood samples. ROC curve obtained through comparing the SCNV calling results with Droplet Digital PCR results show improvement of our method comparing to existing methods. Conclusions: As a summary, our study has three main results/contributions. First, we developed a novel computational tool for SCNV calling in targeted sequencing of ctDNA, with good sensitivity and specificity. Second, we thoroughly evaluated the performance of current available CNV calling tools in targeted sequenced ctDNA. Third, the raw data generated from our six real samples of gradient mixture of different cell lines and human’s samples can serve as an evaluation standard to other further computational tools for CNV calling in targeted sequenced ctDNA.

Rapid screening of NPS and conventional drugs of abuse (DoA) using a Biochip Array Technology (BAT) in whole blood and urine samples from forensic and clinical cases: A comparative study

To evaluate the multi-analyte BAT versus LC-MSMS for the screening of NPS and conventional DoA on real blood and urines samples from forensic and clinical cases. A total of 77 blood samples (B) (9 clinical and 67 forensic cases: 46 deaths, 17 DUID, 3 DFC, 2 homicides) and 81 urines samples (U) (5 forensic: 4 deaths, 1 drugs possession, and 75 clinical: 21 drugs abuse, 18 suspected poisonings, 25 workplace drug testing WPDT, 12 clinical investigation) were screened on Evidence MultiSTAT (Randox ® ) for: alpha-PVP (Cut-off: (B): 5 ng/mL; (U): 5 ng/mL), JWH18 (only U: 20), UR-144 (only U: 10) and AB PINACA (2; 2) derivatives, fentanyls (1; 2), benzoylecgonine BZE (25; 150 ng/mL), amphetamine (50; 200), methamphetamine (50; 200), THC (10; 20), opiates (80; 200), oxycodone (10; 50), 6-MAM (10; 10), methadone (10; 300), buprenorphine (2; 1), benzodiazepines (20; 150), tricyclic antidepressants (60; 150), barbiturates (50; 200), tramadol (5; 5) and pregabalin (only B: 1000). 200 μL of urine or a diluted blood (1:4) were centrifuged, spiked into the biochip and loaded into the system, the remaining process is fully automated and allows to image the sample signal against a cut-off material in 17 min. Results were compared to LC-MSMS and/or LCHRMS screenings. The sensitivity (false negative FN) and specificity (false positive FP) of BAT was calculated based on mass spectrometry cut-offs. BAT assay has allowed the detection of newly marketed NPS in urine like αPHP, MDPHP, pentylone, pentedrone and hexedrone ( n = 4). As alpha-PVP panel does not include the detection of mephedrone derivatives, 3 of them (mephedrone, 3-MMC and 4-MEC) were not detected in 4 urines samples. No blood sample was tested positive for alpha-PVP. Screening of synthetic cannabinoids was also negative in both BAT and LC-MSMS experiments. Tow fentanyls derivatives (3-fluorofentanyl at 3.3 ng/mL and ocfentanyl at 284 ng/mL) and one synthetic benzodiazepines (etizolam at 125 ng/mL) were detected in urine. THC screening presents a good sensitivity/specificity ( n = 44, 17B, 27U). 2 blood samples with low concentrations (0.1 to 2 ng/mL) were tested negative by MultiSTAT. BZE was found in 29 samples (14B, 16U), one FN in urine was observed (BZE at 18 ng/mL). A total of 53 samples (31B, 22U) were tested positive for opiates, 6 were missing from BAT assay in blood (concentrations < 44 ng/mL). No FN was detected. Oxycodone was detected in 4 cases (1B, 3U), but 7 FP were observed in urine due to the presence of codeine and morphine at high concentrations. 6-MAM panel allows detecting heroin intake. 2 urines were screened positive, one at a concentration as low as 10 ng/mL. Methadone was detected in 10 samples (5B, 5U) and 10 samples (4B, 6U) were screened positive for buprenorphine, with 2 FN on MultiSTAT due to a lack of sensitivity (concentration < 1.8 ng/mL). Pregabalin and tramadol, drugs that are increasingly abused, were successfully detected in blood ( n = 3 and 12, respectively). In urine, 6 FP occurred for tramadol due to a cross-reaction with venlafaxine ( n = 4, concentration > 2000 ng/mL) or some alpha-PVP derivatives ( n = 2). Screening of benzodiazepines ( n = 55), TCA ( n = 5), and barbiturates ( n = 2) was satisfactory. Low sensitivity (8 FN, concentration range: 55 to 160 ng/mL) and specificity (19 FP) of methamphetamine/amphetamine panels should be addressed with regard to the interference of some putrefactive bases like β-phenylethylamine, formed in post-mortem samples. This is a promising screening approach that allows the detection of a wide panel of DoA and designer drugs in a variety of matrices including postmortem blood. The limitation of some tests should be addressed and improved.

Bifunctional and highly sensitive electrochemical non-enzymatic glucose and hydrogen peroxide biosensor based on NiCo2O4 nanoflowers decorated 3D nitrogen doped holey graphene hydrogel

In this work, a simple strategy for fabricating a 3D nitrogen doped holey graphene hydrogel decorated with NiCo2O4 nanoflowers (NHGH/NiCo2O4) via a one-pot hydrothermal method with subsequent calcination is reported for the first time. The novel NHGH/NiCo2O4 nanocomposites featured high electrical conductivity, large and accessible surface areas, abundant active sites, and excellent electrocatalytic performance. Considering the excellent catalytic activity of NiCo2O4, a sensitive and bifunctional electrochemical non-enzymatic biosensor was established for the determination of glucose and hydrogen peroxide (H2O2). The obtained biosensor exhibited wide linear ranges (glucose: 0.005–10.95 mM; H2O2: 1–510 μM) and a low detection limits (glucose: 0.39 μM; H2O2: 0.136 μM) in alkaline solution (S/N = 3). Excellent electrocatalytic activity of this sensor was ascribed to the synergistic effects of the hybrid structure between the NiCo2O4 nanoflowers and NHGH. Furthermore, the sensitive biosensor also exhibited high selectivity and could be applied to determine glucose in real blood samples. Taken together, the results reveal that the proposed hybrid nanocomposite could be a promising electrochemical biosensor.