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The unbiased selection of peptide precursors makes data-independent acquisition (DIA) an advantageous alternative to data-dependent acquisition (DDA) for discovery proteomics, but traditional multiplexed quantification approaches employing mass difference labeling or isobaric tagging are incompatible with DIA. Here, we describe a strategy that permits multiplexed quantification by DIA using mass defect-based N,N-dimethyl leucine (mdDiLeu) tags and high-resolution tandem mass spectrometry (MS(2)) analysis. Millidalton mass differences between mdDiLeu isotopologues produce fragment ion multiplet peaks separated in mass by as little as 5.8 mDa, enabling up to 4-plex quantification in DIA MS(2) spectra. Quantitative analysis of yeast samples displayed comparable accuracy and precision for MS(2)-based DIA and MS(1)-based DDA methods. Multiplexed DIA analysis of cerebrospinal fluid revealed the dynamic proteome changes in Alzheimer’s disease, demonstrating its utility for discovery of potential clinical biomarkers. We show that the mdDiLeu tagging approach for multiplexed DIA is a viable methodology for investigating proteome changes, particularly for low-abundance proteins, in different biological matrices.

The zeolitic imidazolate framework (ZIF-8), composed of zinc ion and dimethylimidazole, is widely used in drug delivery because of the easy fabrication process and the good biosafety. However, ZIF-8 suffers from low affinity to nonelectric-rich drugs and does not have surface functional groups. Here, to deliver doxorubicin (DOX) with ZIF-8 to specific target sites, DOX was first modified with a pH-sensitive linker containing two carboxyl groups to form the inactive prodrug CAD and subsequently seeded inside ZIF-8 by a 5 min mineralization process. CAD has high affinity to ZIF-8 because of the carboxyl groups and can anchor to the ZIF-8 surface to enable the surface modification with folic acid for tumor targeting. Moreover, the DOX release is precisely controlled by three steps of acidic pH response, with the dissociation of the FA layer, the breakdown of the ZIF-8 structure, and the cleavage of the pH-sensitive linker in prodrug. This novel "prodrug-ZIF-8" strategy has opened a new horizon in drug delivery.

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolutions and 20 pixels/s throughout molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of 2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high-spatial-resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.

Targeted mass spectrometry (MS) is an important measurement approach in metabolomics with strong analytical performance, given its specificity, sensitivity, and quantitative capacity. However, traditional targeted-MS relies heavily on chemical standards for the development of various detection panels; thus, its metabolite coverage is often limited to those well-known and commercially available compounds. To address this fundamental gap, we previously developed a novel approach [ H. Gu et al. Anal. Chem. 2015 , 87 , 12355 - 12362 ], globally optimized targeted (GOT)-MS, which enables reliable metabolic analysis with broad coverage using a single triple quadrupole instrument. In the present study, we further developed and optimized an innovative targeted MS approach, database-assisted globally optimized targeted (dGOT)-MS, which utilizes the HMDB and METLIN databases to significantly improve both identification and metabolite coverage. As it is well-known, these metabolomics databases have a comprehensive collection of metabolites and their tandem MS spectra; therefore, in this study, multiple reaction monitoring transitions (MRMs) were directly obtained from the databases and, after optimizing MS parameters for those MRMs, 927 metabolites were measured from a plasma aqueous extract sample with high reliability by dGOT-MS. Of these, 310 were confirmed using pure chemical standards while the rest were annotated by identification level using database entries. Furthermore, using breast cancer diagnosis as a proof-of-principle metabolomics application, we showed dGOT-MS to significantly outperform a traditional large-scale targeted MS assay for potential biomarker discovery. In principle, dGOT-MS is able to cover all metabolites (including lipids) that have been characterized in these comprehensive metabolomics databases from various types of biological samples. Therefore, dGOT-MS opens a novel avenue for MS measurements and may play an important role in many future metabolomics studies.

Detection of small metabolites is essential for monitoring and optimizing biological gas conversion. Currently, such detection is typically done by liquid chromatography with offline sampling. However, this method often requires large equipment with multiple separation columns and is at risk of serious microbial contamination during sampling. Here we propose real-time optical detection of small metabolites using uniform plasmonic nanoparticles monolayers produced by capillary-assisted transfer. We reproducibly fabricate metal nanoparticles monolayers with a diameter of ∼1 mm for the detection of acetate, butyrate, and glucose by a glass capillary tube. Metal nanoparticles monolayers are not only uniform in terms of average interparticle distance but also structurally stable under dynamic fluidic conditions. The monolayers resistant to fluid shear stress with surface-enhanced Raman scattering are able to reversibly monitor the concentration of acetate and sensitively detect acetate and glucose at levels as low as 10 μM, which is more than 2 orders of magnitude lower than the concentration range of typical biological gas conversion. In addition, structurally similar metabolites such as acetate and butyrate, when mixed, become distinguishable by our method.

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry dataset. The method enables an automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of family of homologous compounds from MSI images. We show that the KMD filtering, based on an in-house software, is suitable and robust for high resolution (full width at half-maximum, FWHM, at m/z 410 of 20 000) and very high-resolution (FWHM, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria co-cultures and brain tissue section

Stable isotope labeling of peptides is the basis for numerous mass-spectrometry-based quantification strategies. Isobaric tagging and metabolic labeling, namely, tandem mass tagging (TMT) and SILAC, are among the most widely used techniques for relative protein quantification. Here we report an alternative, precursor-based quantification method using nonisobaric TMT variants: TMTzero (TMT0) and superheavy TMT (shTMT). We term this strategy mass difference tandem mass tagging (mTMT). These TMT variants differ by 11 mass units; however, peptides labeled with these reagents coelute, analogous to SILAC-labeled peptide pairs. As a proof-of-concept, we profiled the proteomes of two cell lines that are frequently used in neuroscience studies, SH-SY5Y and SVGp12, using mTMT and standard SILAC-labeling approaches. We show similar quantified proteins and peptides for each method, with highly correlated fold-changes between workflows. We conclude that mTMT is a suitable alternative for precursor-based protein quantification.

Detection of biomarkers in body fluids is critical to both diagnosing the life-threatening diseases and optimizing therapeutic interventions. We herein report use of liquid crystals (LCs) to detect biomarkers in blood with high sensitivity and specificity by employing in situ rolling circle amplification (RCA) on magnetic beads (MBs). Specific recognition of cancer biomarkers, such as platelet derived growth factor BB (PDGF-BB) and adenosine, by aptamers leads to formation of a nucleic acid circle on MBs preassembled with ligation DNA, linear padlock DNA, and aptamers, thereby triggering in situ RCA. LCs change from dark to bright appearance after the in situ RCA products being transferred onto the LC interface decorated with octadecy trimethylammonium bromide (OTAB), which is particularly sensitive to the amplified DNA on MBs. Overall, this label-free approach takes advantages of high specificity of aptamer-based assay, efficient enrichment of signaling molecules on MBs, remarkable DNA elongation performance of the RCA reaction, and high sensitivity of LC-based assay. It successfully eliminates the matrix interference on the LC-based sensors and thus achieves at least 4 orders of magnitude improvement in sensitivity for detection of biomarkers compared to other LC-based sensors. In addition, performance of the developed sensor is comparable to that of the commercial ones. Thus, this study provides a simple, powerful, and promising approach to facilitate highly sensitive, specific, and label-free detection of biomarkers in body fluids.

A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension (1D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension (2D). Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on-off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

Screening and identifying unknown erectile dysfunction (ED) drugs and analogues, which are often illicitly added to health supplements, is a challenging analytical task. The analytical technique most commonly used for this purpose, liquid chromatography-tandem mass spectrometry (LC-MS/MS), is based on the strategy of searching the LC-MS/MS spectra of target compounds against database spectra. However, such a strategy cannot be applied to unknown ED drugs and analogues. To overcome this dilemma, we have constructed a standalone software named AI-SIDA (artificial intelligence screener of illicit drugs and analogues). AI-SIDA consists of three layers: LC-MS/MS viewer, AI classifier, and Identifier. In the second AI classifier layer, an artificial neural network (ANN) classification model, which was constructed by training 149 LC-MS/MS spectra (including 27 sildenafil-type, 6 vardenafil-type, 11 tadalafil-type ED drugs/analogues and other 105 compounds), is included to classify the LC-MS/MS spectra of the query compound into four categories: i.e., sildenafil, vardenafil, and tadalafil families and non-ED compounds. This ANN model was found to show 100% classification accuracy for the 187 LC-MS/MS modeling and test data sets. In the third Identifier layer, three search algorithms (pick-count scoring, simple similarity search, and hybrid similarity search) are implemented. In particular, the hybrid similarity search was found to be very powerful in identifying unknown ED drugs/analogues with a single modification from the library ED drugs/analogues. Altogether, the AI-SIDA software provides a very useful and powerful platform for screening unknown ED drugs and analogues.

Droplet microfluidics enables high-throughput manipulation of fL-μL volume samples. Methods implemented for the chemical analysis of microfluidic droplets have been limited in scope, leaving some applications of droplet microfluidics difficult to perform or out of reach entirely. Nanoelectrospray ionization-mass spectrometry (nESI-MS) is an attractive approach for droplet analysis, because it allows rapid, label-free, information-rich analysis with high mass sensitivity and resistance to matrix effects. Previous proof-of-concept systems for the nESI-MS analysis of droplets have been limited by the microfluidics used so that stable, long-term operation needed for high-throughput applications has not been demonstrated. We describe a platform for the stable analysis of microfluidic droplet samples by nESI-MS. Continuous infusion of droplets to an nESI emitter was demonstrated for as long as 2.5 h, corresponding to analysis of over 20 000 samples. Stable signal was observed for droplets as small as 65 pL and for throughputs as high as 10 droplets/s. A linear-concentration-based response and sample-to-sample carryover of3% were also shown. The system is demonstrated for measuring products of in-droplet enzymatic reactions.

Peroxynitrite (ONOO–) is attracting increasing attention due to its involvement in multiple facets of pathophysiological processes. However, ONOO– bioimaging is still challenging due to (1) the lac...

The 3D cell spheroid is an emerging tool that allows better recapitulating of in vivo scenarios with multiple factors such as tissue-like morphology and membrane protein expression that intimately coordinates with enzyme activity, thus providing a psychological environment for tumorigenesis study. For analyzing different spheroids, conventional optical imaging may be hampered by the need for fluorescent labeling, which could cause toxicity side effects. As an alternative approach, scanning electrochemical microscopy (SECM) enables label-free imaging. However, SECM for cell spheroid imaging is currently suffering from incapability of systematically analyzing the cell aggregates from spheroid generation, electrochemical signal gaining, and the gene expression on different individual cell spheroids. Herein, we developed a top-removable microfluidic device for cell aggregate yielding and SECM imaging methodology to analyze heterotypic 3D cell spheroids on a single device. This technique allows not only on-chip culturing of cell aggregates but also SECM imaging of the spheroids after opening the chip and subsequent qPCR assay of corresponding clusters. Through employment of the micropit arrays (85 × 4) with a top withdrawable microfluidic layer, uniformly sized breast tumor cell and fibroblast spheroids can be simultaneously produced on a single device. By leveraging voltage-switching mode SECM at different potentials of dual mediators, we evaluated alkaline phosphatase without disturbance of substrate morphology for distinguishing the tumor aggregates from stroma. Moreover, this method also enables gene expression profiling on individual tumor or stromal spheroids. Therefore, this new strategy can seamlessly bridge SECM measurements and molecular biological analysis.

Glycans on specific receptors play a crucial role in regulating receptor functions and indicating cell pathological states. For a detailed glycosylation regulatory mechanism, live cell imaging of receptor-specific glycans becomes significantly important. In this work, we present a nongenetically encoded labeling strategy to specifically install a fluorescence resonance energy transfer (FRET) pair onto the receptor of interest by ligand-receptor binding and metabolic glycan engineering. This method breaks the limitation that the receptors have to possess an extracellular terminus which can be used to attach a fluorescent tag, avoiding the undesired effects introduced by inserting amino acids into proteins. Furthermore, the donor-equipped ligand can be flexibly competed with an unlabeled compound, leading to an efficient erasure of donor fluorescence signal. We envision that this strategy will have the potential to sequentially identify and characterize multiple receptor-specific glycans on the live cell surface through multiple cycles including labeled ligand binding, FRET-induced fluorescence imaging, and the unlabeled compound competing for fluorescence erasure. Besides, this in situ glycan profiling strategy will have wide applications in molecular diagnosis and cellular targeted therapies.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsa-stained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.

Thiophenol is the simplest aromatic thiol that is utilized for various applications in industry and agriculture. However, it should be used with care because thiophenol is readily absorbed into the human body by inhalation and ingestion, which leads to serious internal injuries. Thus, there is an urgent need for real-time and accurate monitoring of thiophenol. Despite remarkable advantages of electrogenerated chemiluminescence (ECL) analysis, ECL thiophenol probes have never been reported. Herein, a new strategy for the rapid detection of thiophenol by use of an ECL turn-on chemodosimeter based on a cyclometalated Ir(III) complex is described. This analytical system showed superior sensitivity [limit of detection (LOD) value, 3.8 nM] in comparison to the conventional fluorescence method. In addition, our system exhibited remarkable selectivity and reaction rate toward thiophenol over other analytes. Moreover, it was successfully applied to quantify thiophenol in real water samples, providing a new proof-of-concept for field monitoring based on ECL.

Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying target analytes in clinical and research laboratories. One of the main drawbacks of ELISA is the involvement of multiple washing steps that desorbs the capture antigen/antibody off the polystyrene plate, thereby producing inconsistent and erroneous data. To overcome the problem of desorption, we hypothesized that gelatin nanoparticles (GelNP) could serve as a "plate-adherent" substrate to irreversibly adhere the capture antigen/antibody of interest. We tested our hypothesis using GelNP-based substrate (Gel-BSA-OHG) to adhere 8-hydroxy-2'-deoxyguanosine (8-OHdG) to the polystyrene plate and assayed this molecule using the ELISA technique. The stability and ELISA performance of Gel-BSA-OHG was evaluated in comparison to the conventional substrate (BSA-OHG). Importantly, the Gel-BSA-OHG substrate was found to be more wash-resistant and consequently resulted in improved sensitivity, accuracy, and precision in the ELISA analysis of 8-OHdG. Finally, the scope of Gel-BSA-OHG substrate-based ELISA for clinical application was demonstrated by validating its ability to detect 8-OHdG in an artificial urine sample with high specificity.

An automatic approach to identification of natural products (NPid) in complex extracts by exploring pure shift HSQC (psHSQC) and H2BC spectra of the mixture is developed, which integrated information on chemical shifts (CS), adjacent relationships (AR) and peak intensities (PI) of

As a new analysis tool, photoelectrochemical (PEC) biosensors have been widely studied in recent years. However, common PEC biosensors usually require a highly stable light source to excite the electrical signal and an electrochemical workstation to collect and process the signal data, which limited the development of portable PEC devices. Herein, we propose the design of a sunlight powered portable PEC biosensor that uses sunlight as the light source. The sunlight intensity changes over time and weather and results in varied background PEC currents. To eliminate the interference caused by unstable excitation light, the potentiometric resolve ratiometric principle was introduced. Coupled with a miniature electrochemical workstation and a laptop, a sensitive and portable PEC sensing platform was successfully developed. The detection may be achieved under the irradiation of sunlight and will no longer need an extra light source. In a proof of concept experiment, this platform was successfully applied in aflatoxin B1 analysis, which was promising in the development of portable biosensors.

Not only the degree of acetylation but also the pattern of acetylation of chitosans and chitooligosaccharides (COS) appear to be critical for their biological activities. Protein engineering may expand the toolbox of chitin deacetylases (CDAs) with defined specificities for the enzymatic production of partially deacetylated COS for biotech and biomedical applications. A high-throughput screening (HTS) assay for screening directed evolution libraries is reported. It is based on a fluorescence monitoring assay of the deacetylase activity on COS substrates after capturing the expressed enzyme variants fused to a chitin binding module with chitin-coated magnetic beads. The assay is applied to the screening of random libraries of a Vibrio cholera CDA for increased activity on longer COS substrates.

Multiplex isobaric tags have become valuable tools for high-throughput quantitative analysis of complex biological samples in discovery-based proteomics studies. Hybrid labeling strategies that pair stable isotope mass difference labeling with multiplex isobaric tag-based quantification further facilitate these studies by greatly increasing multiplexing capability. In this work, we present a cost-effective chemical labeling approach that couples duplex stable isotope dimethyl labeling with our custom 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags in a combined precursor isotopic labeling and isobaric tagging (cPILOT) strategy that is compatible with a wide variety of biological samples and permits 24-plex quantification in a single LC-MS/MS experiment. We demonstrate the utility of the DiLeu cPILOT approach by labeling yeast digests and performing proof-of-principle quantification experiments on the Orbitrap Fusion Lumos.

Regardless of major advances in mass spectrometry imaging (MSI), there are three intrinsic limitations associated with MSI, including intricate molecular identification, low molecular coverage, and incapability to obtain "true" spatial distribution due to isobaric and particularly isomeric ions interference. We developed a novel approach that integrates in vivo dual isotope labeling of precursor metabolites with MSI (DLEMMA-MS-Imaging) for identification of spatially localized metabolite and metabolic network map reconstruction. In a proof-of-concept study, we identified 59 and 6 novel metabolites in lemna and tomato fruit, respectively. Significantly, 20-30% of the identified metabolites were found to contain at least one structural isomer that displays a different distribution pattern. The notable feature of this approach is the ability to differentiate localization of structural isomers, hence, providing the "true" distribution of molecules of interest.

A new type of chemiresistor, the impedance-transduced chemiresistor (ITCR), is described for the rapid analysis of glucose. The ITCR exploits porous, high surface area, fluorine-doped carbon nanofibers prepared by electrospinning of fluorinated polymer nanofibers followed by pyrolysis. These nanofibers are functionalized with a boronic acid receptor and stabilized by Nafion to form the ITCR channel for glucose detection. The recognition and binding of glucose by the ITCR is detected by measuring its electrical impedance at a single frequency. The analysis frequency is selected by measuring the signal-to-noise ( S/ N) for glucose detection across 5 orders of magnitude, evaluating both the imaginary and real components of the complex impedance. On the basis of this analysis, an optimal frequency of 13 kHz is selected for glucose detection, yielding an S/ N ratio of 60-100 for [glucose] = 5 mM using the change in the total impedance, Δ Z. The resulting ITCR glucose sensor shows a rapid analysis time (

International audience; Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC50 of 63 μM and an inhibition constant (KI) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.

Carbon nanotube (CNT)-based flexible sensors have been intensively developed for physical sensing. However, great challenges remain in fabricating stretchable CNT films with high electrochemical performance for real-time chemical sensing, due to large sheet resistance of CNT film and further resistance increase caused by separation between each CNT during stretching. Herein, we develop a facile and versatile strategy to construct single-walled carbon nanotubes (SWNTs)-based stretchable and transparent electrochemical sensors, by coating and binding each SWNT with conductive polymer. As a polymer with high conductivity, good electrochemical activity, and biocompatibility, poly(3,4-ethylenedioxythiophene) (PEDOT) acting as a superior conductive coating and binder reduces contact resistance and greatly improves the electrochemical performance of SWNTs film. Furthermore, PEDOT protects the SWNTs junctions from separation during stretching, which endows the sensor with highly mechanical compliance and excellent electrochemical performance during big deformation. These unique features allow real-time monitoring of biochemical signals from mechanically stretched cells. This work represents an important step toward construction of a high performance CNTs-based stretchable electrochemical sensor, therefore broadening the way for stretchable sensors in a diversity of biomedical applications.

Due to exceptional electron-accepting ability, light-absorption, and a delocalized conjugated structure, buckminsterfullerene (C

Currently, there is a severe shortage of donor kidneys that are fit for transplantation, due in part to a lack of adequate viability assessment tools for transplant organs. This work presents the integration of a novel wireless two-channel amperometric potentiostat with microneedle-based glucose and lactate biosensors housed in a 3D printed chip to create a microfluidic biosensing system that is genuinely portable. The wireless potentiostat transmits data via Bluetooth to an Android app running on a tablet. The whole miniaturized system is fully enclosed and can be integrated with microdialysis to allow continuous monitoring of tissue metabolite levels in real time. We have also developed a wireless portable automated calibration platform so that biosensors can be calibrated away from the laboratory and in transit. As a proof of concept, we have demonstrated the use of this portable analysis system to monitor porcine kidneys for the first time from organ retrieval, through warm ischemia, transportation on ice, right through to cold preservation and reperfusion. The portable system is robust and reliable in the challenging conditions of the abattoir and during kidney transportation and can detect clear physiological changes in the organ associated with clinical interventions.

For the first time, simultaneous cathodic and anodic electrochemiluminescence (ECL) emissions of bull serum albumin stabilized Au

The development of practical and sensitive tools for detecting phosphate deficiency could facilitate engineering approaches to enhance crop yield and quality in phosphate-stressed environments, reducing the misuse of nonrenewable fertilizers and their consequent ecological impact. Herein, a 975 nm-activated method based on ZrO

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients.

Plasmonic enzyme-linked immunosorbent assays (ELISA) using the localized surface plasmon resonance (LSPR) of metal nanoparticles has emerged as an appealing alternative to conventional ELISA counterparts for ultrasensitive naked-eye detection of biomolecules and small contaminants. However, batchwise plasmonic ELISA involving end-point detection lacks ruggedness inasmuch as the generation or etching of NP is greatly dependent on every experimental parameter of the analytical workflow. To tackle the above shortcomings, this paper reports on an automatic flow methodology as a reliable detection scheme of hydrogen peroxide related enzymatic bioassays for ultrasensitive detection of small molecules. Here, a competitive ELISA is combined with the in-line generation of plasmonic gold nanoparticles (AuNPs) followed by the real-time monitoring of the NP nucleation and growth rates and size distribution using a USB miniaturized photometer. Glucose oxidase was labeled to the secondary antibody and yielded hydrogen peroxide that acted as the measurand and the reducing agent of the Au(III)/citrate system in the flow network. High-throughput plasmonic assays were feasible by assembling a hybrid flow system composed of two microsyringe pumps, a perfluoroalkoxy alkane reaction coil, and a 26-port multiposition valve and operated under computer-controllable flow conditions. The ultratrace determination of diclofenac in high matrix samples, e.g., seawater, without any prior sample treatment was selected as a proof-of-concept application of the flow-based platform for determination of emerging contaminants via plasmonic ELISA. The detection limit (0.001 μg L-1) was 1 order of magnitude lower than that endorsed by the first EU Watch List for diclofenac as a potentially emerging contaminant in seawater and also than that of a conventional colorimetric ELISA, which in turn is inappropriate for determination of diclofenac in seawater at the levels endorsed by the EU regulation. The proposed automatic fluidic approach is characterized by the reproducible timing in AuNPs nucleation and growth along with the unsupervised LSPR absorbance detection of AuNPs with a dynamic range for diclofenac spanning from 0.01 to 10 μg L-1. Repeatability and intermediate precision (given as normalized signal readouts) in seawater were

Circulating tumor cells (CTCs) are important indicators for tumor diagnosis and tumor metastasis. However, the extremely low levels of CTCs in peripheral blood challenges the precise detection of CTCs. Herein, we report DNA generated electrochemical current combined with rolling circle amplification (RCA) as well as magnetic nanospheres for highly efficient magnetic capture and ultrasensitive detection of CTCs. The antiepithelial cell adhesion molecule (EpCAM) antibody-modified magnetic nanospheres were used to capture and enrich CTCs. The following binding of an aptamer onto the CTC surface and the subsequent RCA assembled a significant amount of DNA molecules onto the electrode. The reaction of the DNA molecules with molybdate can then form redox molybdophosphate and produce an electrochemical current. Using the breast cancer cell MCF-7 as a model, the sensor displays good performances toward detection of MCF-7 that was spiked into peripheral blood. The signal amplification strategy integrated with a magnetic nanosphere platform exhibits good performance in the efficient capture and detection of CTCs, which may find wide potential in cancer diagnostics and therapeutics.

Activatable aptamer probes (AAPs) are promising in molecular imaging of tumors, but the reported shape-switching-dependent AAPs are still challenged by unsatisfied noise suppression, poor stability, and sophisticated sequence design. To address the problem, we constructed a pH-activatable aptamer probe (pH-AAP) by utilizing an acid-labile acetal linker as the responsive element to be fused with a tumor-targeted aptamer. Specifically, a Cy5-labeled aptamer was connected with the quencher BHQ2 through the acetal group, thus generating pH-AAP with quenched fluorescence. Due to the stable proximity of Cy5 to BHQ2, pH-AAP was found to have ultralow background with a quenching efficiency as high as 98%. In comparison with shape-switching-dependent AAPs, the noise suppression of pH-AAP was well maintained for a much longer time in both serum and mouse body, thus showing a robust fluorescence stability. By a combination of the fluorescence recovery induced by acid hydrolysis of acetal linkers and the tumor-targeted recognition of aptamers, pH-AAP could either specifically anchor the extracellular pH-activated signals on the target cell surface in an acidic tumor microenvironment or be activated by acidic lysosomes after it was internalized into target cells. As proof of concept, in vitro evaluation and in vivo imaging of A549 lung cancer cells were performed by using S6 aptamer as a demonstration. It was indicated that pH-AAP realized washing-free, bispecific, and contrast-enhanced tumor imaging. The strategy is simple and free of sequence modification, which promises to provide a universal platform for sensitive and precise tumor diagnosis.

Considerable evidence points to cancer stem-like cells (CSCs) as responsible for promoting progression, metastasis, and drug resistance. Without damage to the cell biological properties, single-cell-derived tumor-sphere is encouraging options for CSCs identification and studies. Although several single cell-based microfluidic methods have been developed for CSCs studies, clarifying liaison between the biomechanics of cells (such as size and deformability) and stem (such as tumor-sphere formation and drug resistance) remains challenging. Herein, we present a platform of integrated microfluidics for the analysis of single-cell-derived tumor-sphere formation and drug resistance. Tumor-spheres derived from different biomechanics (size and/or deformation) single-cells could be formed efficiently using this device. To demonstrate the microfluidic-platform capability, a proof-of-concept experiment was implemented by evaluating single-cell-derived sphere formation of single glioblastoma cells with different biomechanics. Additionally, a course of chemotherapy to study these single-cell-derived spheres was determined by coculture with vincristine. The results indicate that tumor cell biomechanics is associated with single-cell-derived spheres formation; that is, smaller and/or more deformable tumor cells are more stem-like defined by the formation of single-cell-derived spheres than more prominent and/or lesser deformable tumor cells. Also, tumor-spheres derived from single small and/or more deformable tumor cell have higher drug resistance than more prominent and/or less deformable tumor cells. Our device offers a new approach for single-cell-derived sphere formation according to tumor cell different biomechanical properties. Furthermore, it offers a new method for CSC identification and downstream analysis on a single-cell level.

Simultaneous detection of multiple tumor biomarkers is in great demand for early and accurate cancer diagnosis. A homogeneous electrochemical biosensor has been proven to possess high sensitivity, but achieving simultaneous detection of multiple tumor biomarkers is still a challenge. Herein, we develop a novel homogeneous electrochemical biosensor for simultaneous detection of multiple tumor biomarkers based on the functionalized metal-organic frameworks (MOFs). The functionalized MOFs were prepared by using porous UIO-66-NH2 as nanocontainer to load electroactive dyes and dsDNA as a gatekeeper to cap MOFs. In this context, two functionalized MOFs (MB@UIO and TMB@UIO) were fabricated and applied to simultaneous detection of let-7a and miRNA-21, used as the proof-of-concept analytes. The recognition and hybridization of PX with target miRNAs impel the generation of RNA-DNA complexes, which separated from MOFs and allowed the electroactive dyes to be released. In comparison with the case when target miRNAs are absent, two stronger signals are recorded, and dependent on target miRNA concentrations. Thus, simultaneous detection of let-7a and minRNA-21 is achieved, with detection limits down to 3.6 and 8.2 fM, respectively, comparable or lower than those of reported strategies that concentrated on single miRNA detection. Moreover, the proposed biosensor has also been successfully applied for simultaneous detection of target miRNAs spiked in serum samples. Therefore, the proposed strategy was expected to provide more information for early and accurate cancer diagnosis and was an useful application in disease diagnosis and clinical biomedicine.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Molecular analysis of DNA samples with limited quantities can be challenging. Repeatedly sequencing the original DNA molecules from a given sample would overcome many issues related to accurate genetic analysis and mitigate issues with processing small amounts of DNA analyte. Moreover, an iterative, replicated analysis of the same DNA molecule has the potential to improve genetic characterization. Herein, we demonstrate that the use of "click"-based attachment of DNA sequencing libraries onto an agarose bead support enables repetitive primer extension assays for specific genomic DNA targets such as gene exons. We validated the performance of this assay for evaluating specific genetic alterations in both normal and cancer reference standard DNA samples. We demonstrate the stability of conjugated DNA libraries and related sequencing results over the course of independent serial assays spanning several months from the same set of samples. Finally, we finally applied this method to DNA derived from a tumor sample and demonstrated improved mutation detection accuracy.

Reported is a three-electrode mini-cell for voltammetry in 15 μL solutions. The key device component is a rolled platinum foil of an inverted omega-shaped cross section, which functions as both the electrolyte container and the counter-electrode. The analytical assembly was completed with properly sized working and reference electrodes in the two terminals of the quasi-tubular Pt trough. Its applicability in electrochemical assays of 15 μL solutions was verified by redox mediator voltammetry at graphite and noble metal sensors and by trace lead stripping voltammetry. Real sample analysis was adequate for drug detection in a volunteer's blood, drawn before and 1 or 4 h after ingestion of paracetamol. In line with its known pharmacokinetics, lack of drug as well as drug presence and clearance were proven correctly in the three samples. The mini-cell here is easy to assemble and operate, indefinitely reusable, and offers valuable economy in chemical usage and minimal waste. This is primarily a versatile device for electrochemical laboratory analysis of samples that are available only in small quantities, and cost-effective quantitative screens for expensive high-molecular-weight compounds, products of microsynthesis, physiological microdialysis collections, and finger-prick blood sampling are seen as feasible targets.

The removal of sodium dodecyl sulfate (SDS) in SDS-assisted proteomics with electrospray-ionization-mass-spectrometric (ESI-MS) analysis is an essential step in the analysis. Off-line state-of-the-art sample-preparation strategies can allow 100% removal of DS- and up to 100% peptide recoveries. These strategies, however, are typically laborious and require long analysis times and a complex experimental setup. Here, we developed a simple, membrane-free, electrokinetic, on-line, integrated SDS removal-ESI-MS device that was able to enhance ESI-MS signals of bradykinin and peptides from trypsin-digested bovine serum albumin (BSA) in samples that contained SDS micelles. The significant peptide-signal improvements were contributed by the complete removal of DS- and the enrichment of the peptides in the presence of an electric field. Enrichment was via micelle-to-solvent stacking, initially developed in capillary electrophoresis. Bradykinin percent recovery was 800%, and BSA peptide percent recovery was 87%. Enhancement factors in ESI-MS signals (after and before removal) for selected m/ z values of peptides from the BSA digest were 535-693.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.