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Summary Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used to detect intact whole exosomes, yielding exosomal fingerprints within minutes. This rapid exosome detection approach is proposed as a potential tool for cancer studies. Melanoma, a dangerous form of skin cancer, is investigated as a cancer model. The approach allows classification of melanoma cell lines from the stage level through mathematical analysis of the fingerprints and enables the tracking of protein transfer from parental cells to the secreted exosomes by following up certain fingerprint peaks. Protein identities of exosomal fingerprint peaks were clarified by correlation with top-down and bottom-up proteomics. The protein-assigned fingerprints provide a qualitative and semi-quantitative detection of melanoma biomarkers and help to explore melanoma progression via exosome-mediated intercellular communication. Targeting bloodstream-circulating exosomes, the proposed exosome fingerprinting approach also promises fast detection of melanoma diseases and dynamic monitoring of the disease state with proof of concept in mouse and human.

Inkjet printing of electrochemical sensors using a highly loaded mildly edge‐oxidized graphene nanosheet (EOGN) ink is presented. An ink with 30 mg/mL EOGNs is formulated in a mixture of N‐methyl pyrrolidone and propylene glycol with only 30 min of sonication. The absence of additives, such as polymeric stabilizers or surfactants, circumvents reduced electrochemical activity of coated particles and avoids harsh post‐printing conditions for additive removal. A single light pulse from a xenon flash lamp dries the printed EGON film within a fraction of a second and creates a compact electrode surface. An accurate coverage with only 30.4 μg of EOGNs per printed layer and cm2 is achieved. The EOGN films adhere well to flexible polyimide substrates in aqueous solution. Electrochemical measurements were performed using cyclic voltammetry and differential pulse voltammetry. An all inkjet‐printed three‐electrode living bacterial cell detector is prepared with EOGN working and counter electrodes and silver‐based quasi‐reference electrode. The presence of E. coli in liquid samples is recorded with four electroactive metabolic activity indicators.

SummaryIn response to excess nutrients, white adipose tissue expands by both generating new adipocytes and by upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional genomics screen to identify regulators of adipogenesis in the mouse 3T3-L1 cell model. The pooled screening strategy utilized FACS to isolate populations based on lipid content by gating for fluorescence intensity of the lipophilic, green fluorescent BODIPY 493/503 dye. Additionally, this approach categorized if genes functioned during mitotic expansion or lipogenesis. Cellular mechanisms regulating the rates of protein translation and protein stability were found to be critical for adipogenesis and lipogenesis. These mechanisms were further supported by proteomic analyses, which demonstrated that many changes in protein abundance during 3T3-L1 adipogenesis were not driven by transcription. Within these themes, we illustrate that hypusination is critical for translating adipogenic inducers of mitotic expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity to regulate lipogenesis.

Multi-layer inkjet printing is applied to prepare microchips that consist of up to eight three-electrode sensors comprising a carbon nanotube (CNT) working electrode, a silver/silver chloride quasireference electrode and a CNT counter electrode. Optionally, the sensors contain micro-wells to measure sample volumes as small as 50 μL. The implementation of inkjet printing allows the flexible design and highly reproducible fabrication of batches of various electrode shapes and dimensions. The usage of CNTs as standalone working electrodes results in electroanalytical sensors with reliable and superior electrochemical response compared to tested commercial carbon paste electrodes. The sensors can be used at least up to 20 times, e.g., based on an immunoassay based strategy, or in a disposable way, e.g., when measuring in complex matrices such as blood. A gold ink is applied as alternative working electrode material. The printed sensors can be used with compact multichannel potentiostats that are suitable for point-of-care diagnostics.

A noninvasive electrochemical melanoma detection approach based on using adhesive tapes for collecting and fixing cells from a suspicious skin area and transferring the cells into a scanning electrochemical microscope (SECM) is presented. The adhesive layer collects the cells reproducibly and keeps them well adhered on the tape during experiments in an electrolyte solution. A melanoma biomarker, here the intracellular enzyme tyrosinase (TYR), was imaged on the tape-collected cells without further cell lysing using antibodies that were labeled with horseradish peroxidase (HRP). The HRP labels catalyzed the oxidation of a dissolved redox-active species, which was detected at a soft microelectrode, gently brushed in contact mode over the tape. The melanoma biomarker was first detected on tape-stripped samples with murine melanoma cells of different concentrations. Thereafter, increasing levels of TYR were recorded in cells that were collected from the skin of melanoma mouse models representing three different stages of tumor growth. Additionally, SECM results of tape-stripped different human melanoma cell lines were confirmed by previous studies based on traditionally fixed and permeabilized cells.

Bloodstream infections rank among the most serious causes of morbidity and mortality in hospitalized patients, partly due to the long period (up to one week) required for clinical diagnosis. In this work, we have developed a sensitive method to quickly and accurately identify bacteria in human blood samples by combining optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and efficient immunoaffinity enrichment/separation. A library of bacteria reference mass spectra at different cell numbers was firstly built. Due to a reduced sample spot size, the reference spectra could be obtained from as few as 10 to 10(2) intact bacterial cells. Bacteria in human blood samples were then extracted using antibodies-modified magnetic beads for MS fingerprinting. By comparing the sample spectra with the reference spectra based on a cosine correlation, bacteria with concentrations as low as 500 cells per mL in blood serum and 8000 cells per mL in whole blood were identified. The proposed method was further applied to positive clinical blood cultures (BCs) provided by a local hospital, where Escherichia coli and Staphylococcus aureus were identified. Because of the method's high sensitivity, the BC time required for diagnosis can be greatly reduced. As a proof of concept, whole blood spiked with a low initial concentration (10(2) or 10(3) cells per mL) of bacteria was cultured in commercial BC bottles and analysed by the developed method after different BC times. Bacteria were successfully identified after 4 hours of BC. Therefore, an entire diagnostic process could be accurately accomplished within half a day using the newly developed method, which could facilitate the timely determination of appropriate anti-bacterial therapy and decrease the risk of mortality from bloodstream infections.

Herein, we present the intact cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the fingerprinting of human melanoma cancer cell lines grown on aluminium foil. To perform the MALDI-MS assay, melanoma cells were cultured on a flat and thin foil, which was directly transferred to the target plate of MALDI-MS for analysis. The influence of a wide range of cell fixation protocols (i.e. formalin-based and alcohol-based methods) and MALDI matrices on the obtained characteristic spectra was investigated. For the optimization of the MALDI-MS protocol, the MS fingerprints of the melanoma WM-239 cell line with and without an overexpressed enhanced green fluorescent protein were employed. The fingerprints obtained from WM-239 cells grown on aluminium foil were compared with the intact cell MALDI-MS of the cell pellet and presented higher sensitivity in a high m/z range. The optimized protocol was subsequently applied to characterise melanoma cell lines derived from different cancer stages and allowed identification of unique MS signals that could be used for differentiation between the studied cell lines (i.e. molecular weight equal to 10.0 kDa and 26.1 kDa).

A combination of an immuno-affinity enrichment strategy and sensitive amperometric read-out was implemented in a point-of-care platform intended for bacterial infection analysis. Bacterial cells, selectively captured and enriched from complex matrices through immuno-affinity, were detected by amperometric monitoring of the redox state of metabolic activity indicators, providing species identification and viable-cell quantification. The method was successfully employed for the diagnosis of bacterial infections including antimicrobial susceptibility testing with only several hours of total working time. Immunaffinitätsanreicherung und empfindliches amperometrisches Auslesen wurde in einer Point‐of‐Care‐Plattform zur Analyse bakterieller Infektionen kombiniert. Bakterielle Zellen wurden aus komplexen Matrices durch Immunaffinität selektiv eingefangen und angereichert und durch amperometrisches Untersuchen des Redoxzustands stoffwechselaktiver Indikatoren nachgewiesen. Die Identität der bakteriellen Spezies und die Zelllebensfähigkeit wurden so bestimmt. Die Methode wurde erfolgreich zur Diagnose bakterieller Infektionen verwendet, was die Untersuchung der Antibiotikasensibilität innerhalb von nur wenigen Stunden Gesamtarbeitszeit mit einschloss.

We report on a sensitive electrochemical immunoassay for the prostate specific antigen (PSA). An immunoelectrode was fabricated by coating a glassy carbon electrode with multiwalled carbon nanotubes, poly(dimethyldiallylammonium chloride), CeO2 and PSA antibody (in this order) using the layer-by-layer method. The immunosensor is then placed in a sample solution containing PSA and o-phenylenediamine (OPD). It is found that the CeO2 nanoparticles facilitate the electrochemical oxidation of OPD, and this produces a signal for electrochemical detection of PSA that depends on the concentration of PSA. There is a linear relationship between the decrease in current and the concentration of PSA in the 0.01 to 1,000 pg mL−1 concentration range, and the detection limit is 4 fg mL−1. The assay was successfully applied to the detection of PSA in serum samples. This new differential pulse voltammetric immunoassay is sensitive and acceptably precise, and the fabrication of the electrode is well reproducible.

A novel fluorescent immunosensor was developed based on the use of CuS nanoparticles (CuS NPs) as labels for the highly sensitive detection of human prostate cancer biomarker prostate specific antigen (PSA). In the presence of CuS NPs, the non-fluorescent substrate o-phenylenediamine could be oxidized into the stable fluorescent product 2,3-diamiophenazine at physiological pH. Throughout the reaction, no other oxidizing agents (e.g. hydrogen peroxide) were needed. The relatively mild oxidation conditions made the immunoassay robust, reliable and facile. The proposed immunoassay exhibited high sensitivity and specificity for the detection of PSA. A linear relationship between the fluorescent signals and the concentration of PSA was obtained in the range of 0.5 pg mL(-1) to 50 ng mL(-1), with a detection limit of 0.1 pg mL(-1) (S/N = 3). The proposed fluorescent immunoassay can be used as a promising platform for the detection of a variety of other biomarkers.

A novel microfluidic platform integrated with a flexible PDMS-based electrochemical cytosensor was developed for real-time monitoring of the proliferation and apoptosis of HeLa cells. The PDMS-gold film, which had a conductive smooth surface and was semi-transparent, facilitated electrochemical measurements and optical microscope observations. We observed distinct increases and decreases in peak current intensity, corresponding to cell proliferation in culture medium and apoptosis in the presence of an anticancer drug, respectively. This electrochemical analysis method permitted real-time, label-free monitoring of cell behavior, and the electrochemical results were confirmed with optical microscopy. The flexible microfluidic electrochemical platform presented here is suitable for on-site monitoring of cell behavior in microenvironments.

A novel multianalyte electrochemical immunoassay was developed for ultrasensitive detection of human cardiopathy biomarkers cardiac troponin I (cTnI) and human heart-type fatty-acid-binding protein (FABP) using metal ion functionalized titanium phosphate nanospheres (TiP-metal ion) as labels. The metal ions could be detected directly through square wave voltammetry (SWV) without metal preconcentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. The position and size of the peaks reflected the identity and level of the corresponding antigen. The large amount of metal ions loading on the TiP nanospheres greatly amplified the detection signals, and the good biocompatibility of graphene nanoribbons (GONRs) retained good stability for the sandwich-type immunoassay. The proposed immunoassay exhibited high sensitivity and selectivity for the detection of cTnI and FABP. The linear relationships between electrochemical signals and the concentrations of cTnI and FABP were obtained in the range of 0.05 pg/mL-50 ng/mL and 0.05 pg/mL-50 ng/mL, respectively. The detection limits of cTnI and HIgG were 1 and 3 fg/mL (S/N = 3), respectively. Moreover, the immunoassay accurately detected the concentrations of cTnI and FABP in human serum samples, which were demonstrated to have excellent correlations with the standard enzyme linked immunosorbent assay (ELISA) method. The results suggested that the electrochemical immunoassay would be promising in the point-of-care diagnostics application of clinical screening of acute myocardial infarction (AMI) biomarkers.

Point-of-Care (POC) diagnostics grows currently in importance. Portable devices with electrochemical detection are very attractive for the rapid and low cost identification of diseases, especially when centralized laboratories with well-equipped, automatized analytical setups are either not available or results need to be obtained rapidly on site. There is a need for portable POC devices in developing and emerging countries, but also in developed countries. Electroanalysis, often in combination with immunoassay-based detection, provides sensing platforms with high sensitivity and reproducibility without suffering from optical interferences as optical detection methods can often do. For electroanalytical sensors, the selection and costs of the electrode materials and sensor fabrication strategies play major roles. Such considerations need to be made for the complete development chain from designing a sensor, fabricating and optimizing prototypes until reaching the industrial production scale. We found that inkjet printing, which is a digital and mask-less material deposition technique where picoliter droplets of functional inks are jetted with high precision, is the ideal tool for process development of commercial electroanalytical platforms. Herein, we demonstrate the large-scale fabrication of electrochemical microchips with up to eight parallel 50 µL wells containing each carbon nanotube working and counter electrodes, silver/silver chloride quasi-reference electrodes and a dielectric layer to define accurately the dimensions of the electrodes. The employed carbon nanotubes show a superior electrochemical behaviour and the multi-layer inkjet printing process provides highly reproducible electrode surfaces. We shall demonstrate the application as sensing platforms for antioxidants and hormones in several biological fluids using a portable, in-house made multichannel potentiostat. References [1] http://lepa.epfl.ch/SENSaSION [2] A. Lesch, F. Cortés-Salazar, M. Prudent, J. Delobel, S. Rastgar, N. Lion, J.-D. Tissot, P. Tacchini, H. H. Girault, J. Electroanal. Chem., 2014, 61-68, 717. [3] M. Jović, Y. Zhu, A. Lesch, A. Bondarenko, F. Cortés-Salazar, F. Gumy, Hubert H. Girault, J. Electroanal. Chem., submitted. [4] A. Lesch, F. Cortés-Salazar, V. Amstutz. P. Tacchini, H. H. Girault, Anal. Chem., 2015, 87, 1026. [5] A. Lesch, S. Maye, M. Jović, F. Gumy, P. Tacchini, H. H. Girault, In Biotech, Biomaterials and Biomedical TechConnect Briefs, 2016, 3, Chapter 4: Sensors, Diagnostics & Imaging, 121.

A microfluidic platform to evaluate the expression of multi-glycans on a cell surface was developed using electrochemical impedance spectroscopy (EIS) and optical microscope technique. In the microfluidic channel, four indium tin oxide (ITO) electrodes were modified with three lectins and one passivation agent, respectively, to selectively recognize the corresponding carbohydrate epitopes on the cell surface. The binding of the cells on the electrode array was monitored by the electrochemical impedance to evaluate the expression of cell surface glycans. The excellent optical transparency of ITO electrode permitted the microscopic observation of the cell binding simultaneously to substantiate the impedance measurement. Compared with the individual technology, the double-check mode increased the sensitivity and accuracy of the assay. The experimental results using these two techniques indicated that the cell binding ability decreased in the order WGACon APNA, which was consistent with the expression difference of carbohydrate epitopes on K562 cell surface. The proposed strategy was further used for facile evaluating the variations of glycan expression on living cells in response to drugs. The consumption of cell sample for each sensing interface in the whole experiments is merely 5 × 10(3) cells. This platform offers great promise for cancer-associated glycol-biomarkers screening and further helps cancer diagnosis and treatment.

Porous titanium phosphate (TiP) nanoparticles with high water solubility have been synthesized by using the surfactant sodium dodecyl sulfate (SDS) as the structure-directing agent. The TiP nanoparticles can be functionalized with Cd(2+) and can be further used as labels for electrochemical detection of proteins due to their excellent ion-exchange property.

Herein we present the large scale fabrication of multiplexed three electrode sensors used in a point of care device platform that couples a magnetic bead based immunoassay strategy with amperometric detection for rapid and highly sensitive analysis. The multiplexed sensors consisted of eight independent electrochemical cells each with a carbon nanotube (CNT) working electrode \{CNT\} counter electrode and a silver silver chloride quasi reference electrode. The microchips were fabricated on flexible polyethylene terephthalate (PET) sheets by sequential multilayer inkjet printing (IJP) of silver \{CNT\} and insulator inks that were either simultaneously or subsequently post processed (e.g. through \{UV\} photo polymerization or photonic curing). Finally plastic wells were mounted on top of the inkjet printed patterns to obtain an eight well microtiter plate where each well had a solution capacity of 50 µL. Due to the high precision of the \{IJP\} process the microtiter plates showed high reproducibility among the individual electrochemical cells (1–2 of deviation). Furthermore the microchips can be reusable for at least up to 20 times as demonstrated herein. In a customized multichannel potentiostat with eight implemented magnets matching the positions of the working electrodes the electrochemical readout of magnetic bead based sandwich and competitive immunoassays was successfully realized for the detection of thyroid stimulating hormone (TSH) and atrazine (ATR) in aqueous and urine samples respectively. The achieved limits of detection for \{ATR\} (i.e. 0.01 µg/L) and \{TSH\} (i.e. 0.5 µIU/mL) demonstrated the potential of the \{IJP\} microtiter plates for the environmental and biological quantification of analytes in a very reliable high throughput platform. This work shows that \{IJP\} has certainly reached the status of a batch production tool for electroanalytical sensing platforms.

Titanium dioxide-modified target plates were developed to enhance intact bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plates were designed to photocatalytically destroy the bacterial envelope structure and improve the ionization efficiency of intracellular components, thereby promoting the measurable mass range and the achievable detection sensitivity. Accordingly, a method for rapid detection of antimicrobial resistance-associated proteins, conferring bacterial resistance against antimicrobial drugs, was established by mass spectrometric fingerprinting of intact bacteria without the need for any sample pre-treatment. With this method, the variations in resistance proteins' expression levels within bacteria were quickly measured from the relative peak intensities. This approach of resistance protein detection directly from intact bacteria by mass spectrometry is useful for fast discrimination of antimicrobial-resistant bacteria from their non-resistant counterparts whilst performing species identification. Also, it could be used as a rapid and convenient way for initial determination of the underlying resistance mechanisms.

Skin problems are often overlooked due to a lack of robust and patient-friendly monitoring tools. Herein, we report a rapid, noninvasive, and high-throughput analytical chemical methodology, aiming at real-time monitoring of skin conditions and early detection of skin disorders. Within this methodology, adhesive sampling and laser desorption ionization mass spectrometry are coordinated to record skin surface molecular mass in minutes. Automated result interpretation is achieved by data learning, using similarity scoring and machine learning algorithms. Feasibility of the methodology has been demonstrated after testing a total of 117 healthy, benign-disordered, or malignant-disordered skins. Remarkably, skin malignancy, using melanoma as a proof of concept, was detected with 100% accuracy already at early stages when the lesions were submillimeter-sized, far beyond the detection limit of most existing noninvasive diagnosis tools. Moreover, the malignancy development over time has also been monitored successfully, showing the potential to predict skin disorder progression. Capable of detecting skin alterations at the molecular level in a nonsurgical and time-saving manner, this analytical chemistry platform is promising to build personalized skin care.