Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples.

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform.

As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (µM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.

Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction.

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.

Ultrasensitive Electrochemical Biosensor Developed by Probe Lengthening for Detection of Genomic DNA in Human Serum.

As an alternative to most of the reported nucleic acid amplification-based electrochemical DNA biosensors used for detection of trace levels of genomic DNA, we herein present a novel detection concept. The proposed system involves the conversion of two short double-stranded DNAs (dsDNAs), labeled with a thiol-tag or biotin-tag, into a single integrated dsDNA containing thiol and biotin at both terminals in the presence of target DNA through ligase chain reaction (LCR) and followed by the immobilization of these integrated dsDNAs on a bovine serum albumin (BSA)-modified gold electrode surface. Owing to rapid depletion of the two short dsDNAs via LCR, the integrated dsDNAs were generated in an exponential manner so that this sensoring approach offered a limit of detection of 25 yoctomoles (15 copies in 50 µL sample volumes), a high discrimination of single-base mismatch and a wide linear concentration range (across 6 orders of magnitude) for target DNA. Significantly, the proposed sensor, which has simplicity in operation and ease of miniaturization, detected the target of interest in total nucleic acid extracts derived from clinical serum samples with excellent results, thereby demonstrating its considerable diagnostic potential in fields ranging from virus detection to the diagnosis of genetic diseases.

Improving quantitative control and homogeneous distribution of samples on paper-based analytical devices via drop-on-demand inkjet printing.

A standard desktop printer with multiple ink cartridges can accurately deposit a broad variety of biomaterials on microfluidic paper-based analytical devices (µPADs) which have been extensively applied to environmental monitoring and screening of food and beverage contamination. Finding ways to realize sample quantitative control by tuning the CMYK value, however, remains challenging. Herein, we studied the influence of the CMYK value on the ink volume jetted by ink cartridges. The regularity research on a single-color and two-colors was performed in two print mode-grayscale printing and color printing. The results demonstrated that the number of ink dots increased with the increase of the gray value and opacity value, which means that the amount of the bio-ink increases with the increase of the CMYK value. The 3,3',5,5'-tetramethylbenzidine-horseradish peroxidase-hydrogen peroxide, glucose oxidase-horseradish peroxidase and bull serum albumin-citrate buffer-tetrabromophenol blue systems were chosen as examples to prove the print regularity. Samples and assay reagents can be quantitatively deposited on a substrate by adjusting the CMYK value with as many as four ink cartridges. The present approach has been successfully applied to assay the targets in real serum samples, showing the potential application of the most common office piezoelectric printer in µPADs.

Glomerular Filtration Rate Is a Major Predictor of Clearance of Oxcarbazepine Active Metabolite in Adult Chinese Epileptic Patients: A Population Pharmacokinetic Analysis.

BACKGROUND: Oxcarbazepine (OXC) is almost completely metabolized to its10-monohydroxy derivative (MHD), which is responsible for the pharmacological effects of the drug. Several studies have described the population pharmacokinetics (PPK) of MHD in pediatric patients, but little is known about its pharmacokinetics in adult patients. In addition, no study to date has proposed a model to investigate the influence of genetic polymorphisms on MHD pharmacokinetics. The aim of this study was to establish a PPK model of MHD to investigate the effects of genetic polymorphisms in UGT2B7, UGT1A9, ABCB1, and ABCB2 in adult Chinese patients with epilepsy and to develop a new dosage guideline for OXC. METHODS: Data were prospectively collected from 187 adult patients with epilepsy who were taking OXC. MHD trough concentrations were detected by enzyme-multiplied immunoassay. Patients were genotyped for 4 single nucleotide polymorphisms (UGT2B7 802T>C, UGT1A9 I399C>T, ABCB1 3435C>T, and ABCB2 1249G>A). Other covariates included sex, age, body weight (BW), hepato-renal function, and concomitant medications. Data were analyzed using the nonlinear mixed effects modelling software. RESULTS: The apparent clearance (CL) of MHD was significantly influenced by glomerular filtration rate and BW, and was unrelated to other covariates such as genetic polymorphisms and coadministration with levetiracetam, lamotrigine, and topiramate. Moreover, a new dosage guideline was proposed based on the final model to individualize OXC regimens for adult patients with varying BW and renal function. CONCLUSIONS: Glomerular filtration rate was first found as an important covariate influencing MHD CL. A PPK model was established to estimate the individual MHD CL for adult patients taking OXC and may be applied for individualizing doses in the target population.

Population pharmacokinetics of oxcarbazepine active metabolite in Chinese paediatric epilepsy patients and its application in individualised dosage regimens.

PURPOSE: Oxcarbazepine (OXC) is an antiepileptic drug metabolised to active 10-monohydroxy derivative (MHD) following oral administration. There are no MHD population pharmacokinetic (PPK) models that describe the influence of genetic factors on MHD pharmacokinetics (PK). We developed a PPK model of MHD to investigate gene polymorphism of enzymes associated with MHD PK in Chinese paediatric epilepsy patients and evaluated its utility for dose individualisation. METHODS: Data were prospectively collected from 141 paediatric epilepsy patients (aged ≤ 14 years) who received OXC therapy at the First Affiliated Hospital of Fujian Medical University. The trough concentrations at steady state were determined by enzyme-multiplied immunoassay. Patients were genotyped for four single nucleotide polymorphisms (UGT2B7 802T>C, UGT1A9 I399C>T, ABCB1 3435C>T, and ABCB2 1249G>A). Patient gender, age, body weight (BW), hepatorenal function, and co-administrations were recorded. The PPK model was developed using nonlinear mixed-effects modelling software. The clinical performance of the final model was evaluated by including additional paediatric patients (n = 20) in the validation group. RESULTS: Oral clearance of MHD was significantly influenced by BW. The MHD PK was unrelated to the other covariates, such as the four single nucleotide polymorphisms and co-administration with new-generation antiepileptic drugs. The final BW-dependent exponent model showed the best fit with our data and predicted the trough concentrations in the validation group more accurately than the basic model. A new dosing strategy combining the dosage guideline and Bayesian method is proposed to individualise OXC regimens. CONCLUSION: A PPK model was established to estimate individual MHD clearance in paediatric patients taking OXC to develop individualised OXC dosing regimens for Chinese paediatric epilepsy patients.

Platinum nanoparticles/graphene-oxide hybrid with excellent peroxidase-like activity and its application for cysteine detection.

A facile approach is proposed for the growth of platinum nanoparticles on graphene oxide (PtNPs/GO). The resulting PtNPs/GO hybrid has been proved to function as peroxidase mimics that can catalyze the oxidation of peroxidase substrates in the presence of hydrogen peroxide (H2O2). Kinetic studies indicate that the PtNPs/GO nanocomposite has a considerably higher affinity for both 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 than those of other platinum-based peroxidase mimics. Furthermore, colorimetric recognition and sensing of L-cysteine with high sensitivity and selectivity is presented based on target-induced shielding against the peroxidase-like activity of PtNPs/GO. We envision that this material will be an ideal candidate for a wide range of potential applications in the fields of biomedicine and environmental chemistry.

Citrate-capped platinum nanoparticle as a smart probe for ultrasensitive mercury sensing.

An easily prepared platinum nanoparticle (PtNP) probe for the sensitive and selective detection of Hg(2+) ions is developed here. The PtNPs with an average size of approximately 2.5 nm were prepared by a reduction method with sodium borohydride and trisodium citrate serving as reductant and stabilizer, respectively. The resulting PtNPs could catalyze the reduction of Hg(2+) by surface-capping citrate. The effect of Hg(2+) uptake implies amalgam formation, which leads to remarkable inhibition of the peroxidase-like activity of citrate-capped PtNPs. On the basis of this effect, a colorimetric mercury sensor was established through the use of citrate-capped PtNPs to catalyze the colorimetric system of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2. The high specificity of the Hg-Pt interaction provides the excellent selectivity for Hg(2+) over interfering metal ions. The sensitivity of this smart probe to Hg(2+) is extremely excellent with a limit of detection (LOD) as low as 8.5 pM. In view of these advantages, as well as the cost-effectiveness, minimized working steps, and naked-eye observation, we expect that this colorimetric sensor will be a promising candidate for the field detection of toxic Hg(2+) ions in environmental, biological, and food samples.

In situ growth of porous platinum nanoparticles on graphene oxide for colorimetric detection of cancer cells.

A green approach is proposed for in situ growth of porous platinum nanoparticles on graphene oxide (PtNPs/GO). The resulting nanocomposite has been proven to function as peroxidase mimetics that can catalyze the reaction of peroxidase substrate in the presence of hydrogen peroxide. On the basis of the peroxidase-like activity, we used the PtNPs/GO as a signal transducer to develop a colorimetric assay for the direct detection of cancer cells. By using folic acid as a recognition element, a total of 125 cancer cells (MCF-7) can be distinguished by naked-eye observation. We envision that this nanomaterial could be used as a power tool for a wide range of potential applications in biotechnology and medicine.

A homogeneous electrochemiluminescence immunoassay platform based on carbon quantum dots and magnetic beads enrichment for detection of thyroglobulin in serum.

Considering the high probability of recurrence or metastasis after thyroidectomy, it is meaningful to develop a rapid, sensitive and specific method for monitoring thyrophyma-related biomarkers. In this study, a homogeneous electrochemiluminescence immunoassay (HO-ECLIA) coupled with magnetic beads (MBs)-based enrichment tactic was established for the determination of thyrophyma-related thyroglobulin (Tg). Importantly, owing to the abundant surface groups and good biocompatibility of carbon quantum dots (CQDs), the incorporation of CQDs onto the Tg antigen surface was achieved, resulting in the formation of Tg-encapsulated CQDs (CQDs-Tg), which served not only as an ECL probe but as a biorecognition element. Under optimal experimental conditions, the proposed platform demonstrated a wide linear range from 0.01 to 100 ng·mL-1 with a detection limit of 6.9 pg·mL-1 (S/N = 3), and performed well in real serum sample analysis against interference. Collectively, the proposed platform exhibited the rapid response, satisfactory sensitivity and specificity toward Tg in complex serum milieu, and held a considerable potential for clinical prognosis monitoring of thyrophyma.

Bovine serum albumin-based probe carrier platform for electrochemical DNA biosensing.

A bovine serum albumin (BSA)-monolayer-based probe carrier platform is shown to improve the performance of a conventional thiolated single-stranded DNA probe self-assembled-monolayer-based electrochemical DNA hybridization biosensor. A detection limit of 0.5 fM can be obtained in a very reproducible manner (relative standard deviation <5%), along with high specificity. The performance of the biosensor can be attributed primarily to the enhanced spatial positioning range and accessibility of the probes on the BSA-based platform. Furthermore, the novel biosensor shows high resistance to nonspecific adsorption of nucleic acid and protein and can be directly employed in detection in biological fluids. These advantages give this simple developed methodology great promise for a wide range of nucleic acid testing.

An IMPLICATION logic gate based on citrate-capped gold nanoparticles with thiocyanate and iodide as inputs.

Herein we developed an IMPLICATION logic gate based on citrate-capped AuNPs by employing thiocyanate (SCN(-)) and iodide (I(-)) as inputs, and devised a colorimetric sensor for the determination of I(-) with good selectivity and sensitivity. To the best of our knowledge, this is the first example in which two species of anions serve as inputs to obtain visually observed Boolean outputs. Under the optimum conditions, 0.8 µM I(-) could induce a significant color change and be recognized by the naked eye. The detection limit is 50 nM by using UV-vis spectroscopy.

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia.

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a "sandwich" sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-µL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.

Electrochemical immunosensor based on AuNPs/ERGO@CNT nanocomposites by one-step electrochemical co-reduction for sensitive detection of P-glycoprotein in serum.

P-glycoprotein (P-gp), a transmembrane glycoprotein widely expressed on the surface of various cells, is highly associated with multidrug resistance (MDR) that heralds the malignant progress of disease after drug treatment. Notably, there have been reported that serum P-gp is a potential marker for assessing the progression of disease resistance. Currently, there are few methods for point-of-care serum P-gp detection. In this study, we proposed a gold nanoparticles/electrochemically reduced graphene oxide@carbon nanotube (AuNPs/ERGO@CNT) modified immunosensor based on a one-step electrochemical co-reduction method. The limit of detection (LOD) of our constructed electrochemical immunosensor for P-gp detection reached 0.13 ng/mL, and the detection results in serum were consistent with ELISA. The developed immunosensor is expected to provide a scientific basis for the clinical application of serum P-gp monitoring and integrated medicine.

Comparison of the peroxidase-like activity of unmodified, amino-modified, and citrate-capped gold nanoparticles.

The origin of the peroxidase-like activity of gold nanoparticles and the impact of surface modification are studied. Furthermore, some influencing factors, such as fabrication process, redox property of the modifier, and charge property of the substrate, are investigated. Compared to amino-modified or citrate-capped gold nanoparticles, unmodified gold nanoparticles show significantly higher catalytic activity toward peroxidase substrates, that is, the superficial gold atoms are a contributing factor to the observed peroxidase-like activity. The different catalytic activities of amino-modified and citrate-capped gold nanoparticles toward 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) show that the charge characteristics of the nanoparticles and the substrate also play an important role in the catalytic reactions.

Peroxidase-like activity of water-soluble cupric oxide nanoparticles and its analytical application for detection of hydrogen peroxide and glucose.

Water-soluble cupric oxide nanoparticles are fabricated via a quick-precipitation method and used as peroxidase mimetics for ultrasensitive detection of hydrogen peroxide and glucose. The water-soluble CuO nanoparticles show much higher catalytic activity than that of commercial CuO nanoparticles due to their higher affinity to hydrogen peroxide. In addition, the as-prepared CuO nanoparticles are stable over a wide range of pH and temperature. This excellent stability in the form of aqueous colloidal suspensions makes the application of the water-soluble CuO nanoparticles easier in aqueous systems. A colorimetric assay for hydrogen peroxide and glucose has been established based on the catalytic oxidation of phenol coupled with 4-amino-atipyrine by the action of hydrogen peroxide. This analytical platform not only confirms the intrinsic peroxidase-like activity of the water-soluble cupric oxide nanoparticles, but also shows its great potential applications in environmental chemistry, biotechnology and medicine.

Bare gold nanoparticles as facile and sensitive colorimetric probe for melamine detection.

In this study, we demonstrate a more sensitive colorimetric assay for the detection of melamine based on the strong attraction between exocyclic amine groups of melamine and surface-bound AuCl(4)(-)/AuCl(2)(-) ions on bare gold nanoparticles prepared by the borohydride reduction method. This method is rather simple without further modification of gold nanoparticles and complicated pretreatment of samples. The detection limit of the method is 2.0 × 10(-7) g L(-1) for melamine in infant formula, which is much lower (about 1000-fold) than that of using citrate-capped GNPs as a colorimetric probe. The proposed scheme could be a good alternative means for on-site and real-time screening of melamine adulterant in infant formula without costly instruments.

Sequence-specific amperometric detection based on a double-probe mode and enzyme-mediated multiple signal electrocatalysis for the double-stranded DNA of PML/RARα-related fusion gene.

A new electrochemical DNA biosensor based on double-probe mode and enzyme-mediated multiple signal electrocatalysis is constructed for the highly sensitive determination of double-stranded (ds-) PML/RARα fusion gene. Through the ingenious design of two groups of detection probes, including two thiolated capture probes anchored on dual standalone detection units integrated into one customized gold electrode and four biotinylated reporter probes, hybridizing with different segments of the same target single-stranded DNA (ssDNA) simultaneously, the hybridization efficiency between the probes and target is improved by preventing the reannealing of the two separate target ssDNA. Compared with a single reporter probe, this method can dramatically increase the amount of biotin and introduce numerous streptavidin-labelled horseradish peroxidase (HRP), thereby significantly amplifying electrochemical signals with low background signals. The combination of the dual-probe mode, multiple signal amplification strategy, and the inherent electrocatalytic activity of the HRP results in the prominent electrochemical sensing performance in detecting large-fragment target dsDNA with a detection limit as low as 71 fM. Furthermore, taking advantage of the new detection strategy, polymerase chain reaction (PCR) products and enzyme-digested PCR products from NB4 cells can be effectively analysed, showing great promise for the development of a new class of point-of-care platforms for disease-/drug-related genes.

Enhanced Cancer Starvation Therapy Enabled by an Autophagy Inhibitors-Encapsulated Biomimetic ZIF-8 Nanodrug: Disrupting and Harnessing Dual Pro-Survival Autophagic Responses.

Autophagy is an important protective mechanism in maintaining or restoring cell homeostasis under physiological and pathological conditions. Nanoparticles (NPs) with certain components and morphologies can induce autophagic responses in cancer cells, providing a new perspective for establishing cancer therapy strategies. Herein, a novel nanodrug system, cell membranes-coated zeolitic imidazolate framework-8 (ZIF-8) NPs encapsulating chloroquine (CQ) and glucose oxidase (GOx) (defined as mCG@ZIF), is designed to achieve an enhanced anticancer effect with the combination of starvation therapy and an autophagy regulation strategy. It is found that ZIF-8 as a nanocarrier can induce autophagy to promote survival of cancer cells via the upstream Zn2+-stimulated mitochondrial reactive oxygen species (ROS) so that the anticancer effect is directly achieved by inhibiting this pro-survival autophagy using CQ released from mCG@ZIF under a tumor acidic microenvironment. Moreover, a cancer cell under starvation caused by GOx harnesses autophagy to maintain intracellular ATP levels and resist starvation therapy. The released CQ further inhibits the starvation-induced pro-survival autophagy and cuts off the protective pathway of cancer cells, enhancing the anticancer efficiency of GOx-based starvation therapy. Significantly, the cell membrane coating endows mCG@ZIF with excellent in vivo homotypic targeting ability. Both in vitro and in vivo results have confirmed the enhanced anticancer effect achieved by mCG@ZIF with a negligible side effect.