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Self-absorption of spectral lines is known to lower the performance of analytical measurements via calibration-free laser-induced breakdown spectroscopy. However, the error growth due to this effect is not clearly assessed. Here we propose a method to quantify the measurement error due to self-absorption based on the calculation of the spectral radiance of a plasma in local thermodynamic equilibrium. Validated through spectroscopic measurements for a binary alloy thin film of compositional gradient, the method evidences that measurement performance lowering due to self-absorption depends on the spectral shape of the analytical transition and on the intensity measurement method. Thus, line-integrated intensity measurements of Stark broadened lines enable accurate analysis, even at large optical thickness, if line width and plasma size are precisely known. The error growth due to self-absorption is significantly larger for line shapes dominated by Doppler broadening and for line-center intensity measurements. The findings present a significant advance in compositional measurements via calibration-free laser-induced breakdown spectroscopy, as they enable straightforward selection of most appropriate analytical lines.As an important hydrolytic enzyme, abnormal activity of alkaline phosphatase (ALP) is closely associated with a variety of diseases. It has been identified as an important diagnostic indicator for clinical hepatobiliary and bone diseases. Herein, a novel electrochemical sensor based on signal amplification strategy through ring-opening polymerization (ROP) has been developed to assay of ALP activity. First of all, 3-mercaptopropanoic acid (MPA) was employed as a cross-linking agent to attach O-phosphoethanolamine to the electrode surface via amide bond. Then, ALP catalyzed the hydrolysis of phosphate monoester structures to hydroxyl groups, which could initiate ROP reaction. The polymer grafted on the electrode surface contains a large number of ferrocene electroactive molecules, which effectively increased the signal output of the electrochemical sensor and improved the sensitivity of ALP activity detection. Under optimum conditions, this electrochemical sensor rendered a satisfactory linear dependence over the range from 20 to 120 mU mL-1, with a low detection limit of 0.66 mU mL-1. Furthermore, this strategy presented satisfactory selectivity and interference resistance in human serum sample, and compared with clinical data, the relative error of the results obtained by this method was less than 5%. Thus, this method showed considerable potential for the detection of ALP activity in clinical application.Recombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a critical quality attribute. Thus, analytical methods for profiling EPO glycosylation are highly demanded. Owing to the complexity of the intact protein, information about EPO glycosylation is commonly derived from released glycan and glycopeptide analysis using mass spectrometry (MS). Alternatively, comprehensive insights into the glycoform heterogeneity of intact EPO are obtained using ESI MS-based methods with or without upfront separation of EPO glycoforms. MALDI MS, typically performed with TOF mass analyzers, has been also used for the analysis of intact EPO but, due to the poor glycoform resolution, has only provided limited glycoform information. Here, we present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO with improved glycoform resolution and without loss of sialic acid residues commonly observed in MALDI analysis. Three EPO variants were characterized in-depth and up to 199 glycoform compositions were assigned from the evaluation of doubly-charged ions, without any deconvolution of the mass spectra. Key glycosylation features such as sialylation, acetylation, and N-acetyllactosamine repeats were determined and found to agree with previously reported data obtained from orthogonal analyses. The developed method allowed for a fast and straightforward data acquisition and evaluation and can be potentially used for the high-throughput comparison of EPO samples throughout its manufacturing process.Nanoplasmonic biosensing shows an immense potential to satisfy the needs of the global health industry - low-cost, fast, and portable automated systems; highly sensitive and real-time detection; multiplexing and miniaturization. In this review, we presented the theory of nanoplasmonic biosensing for popular detection schemes - SPR, LSPR, and EOT - and underline the consideration for nanostructure design, material selection, and their effects on refractometric sensing performance. Later, we covered the bottom-up and top-down nanofabrication methods for nanoplasmonic biosensors. Subsequently, we reviewed the recent examples of nanoplasmonic biosensors over a wide range of clinically relevant analytes in the diagnosis and prognosis of a wide range of diseases and conditions such as biomarker proteins, infectious bacteria, viral agents. Finally, we discussed the challenges of nanoplasmonic biosensing toward clinical translation and proposed strategic avenues to be competitive against current clinical detection methods. Hopefully, nanoplasmonic biosensing can realize its potential through successful demonstrations of clinical translation in the upcoming years.CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) proteins are powerful gene-editing tools because of their ability to accurately recognize and manipulate nucleic acids. Besides gene-editing function, they also show great promise in biosensing applications due to the superiority of easy design and precise targeting. To improve the performance of CRISPR/Cas-based biosensing systems, various nucleic acid-based signal amplification techniques are elaborately incorporated. The incorporation of these amplification techniques not only greatly increases the detection sensitivity and specificity, but also extends the detectable target range, as well as makes the use of various signal output modes possible. Therefore, summarizing the use of signal amplification techniques in sensing systems and elucidating their roles in improving sensing performance are very necessary for the development of more superior CRISPR/Cas-based biosensors for various applications. MK8245 In this review, CRISPR/Cas-based biosensors are summarized from two aspects the incorporation of signal amplification techniques in three kinds of CRISPR/Cas-based biosensing systems (Cas9, Cas12 and Cas13-based ones) and the signal output modes used by these biosensors. The challenges and prospects for the future development of CRISPR/Cas-based biosensors are also discussed.Although DNA aptamers can show comparable affinity to antibodies and have the advantage of having high batch-to-batch consistency, they often suffer from unsatisfied specificity for complex samples. The limited library size used for aptamer in vitro isolation (SELEX) has been recognized as one of the major reasons. Programmed cell death-ligand 1 (PD-L1) is both a key protein in cancer diagnostics and also immunotherapy. We report here a DNA aptamer that highly specifically binds PD-L1 expressed on the surface of various cancer cells and multiple types of tissue sections. The aptamers were selected from a DNA library containing a type II restriction endonuclease Alu I recognition site in the middle of the 40-nt random sequences, against recombinant PD-L1 rather than the whole cell or tissue section. The library enrichment was achieved by Alu I mediated-SELEX, named as REase-SELEX, in which Alu I cut off the non-binders at the recognition site and, more importantly, induced library mutations to substantially increase the library diversity. 8-60, a representative aptamer with high affinity (KD = 1.4 nM determined by SPR) successfully detected four types of cancer cells with PD-L1 expression levels from low to high by flow cytometry, normal human tonsil (gold standard for PD-L1 antibody evaluation), clinical non-small cell lung cancer (high PD-L1 expression level), and malignant melanoma (low PD-L1 expression level) tissue sections by fluorescence microscopy imaging, showing unprecedented high specificity. The results demonstrate that 8-60 is an advanced probe for PD-L1 cancer diagnostics and mutations in SELEX greatly favor aptamer specificity.Saliva analysis has been gaining interest as a potential non-invasive source of disease indicative biomarkers due to being a complex biofluid correlating with blood-based constituents on a molecular level. For saliva to cement its usage for analytical applications, it is paramount to gain underpinning molecular knowledge and establish a 'baseline' of the salivary composition in healthy individuals as well as characterize how these factors are impacting its performance as potential analytical biofluid. Here, we have systematically studied the molecular spectral fingerprint of saliva, including the changes associated with gender, age, and time. Via hybrid artificial neural network algorithms and Raman spectroscopy, we have developed a non-destructive molecular profiling approach enabling the assessment of salivary spectral changes yielding the determination of gender and age of the biofluid source. Our classification algorithm successfully identified the gender and age from saliva with high classification accuracy. Discernible spectral molecular 'barcodes' were subsequently constructed for each class and found to primarily stem from amino acid, protein, and lipid changes in saliva. This unique combination of Raman spectroscopy and advanced machine learning techniques lays the platform for a variety of applications in forensics and biosensing.In analytical chemistry spectroscopy is attractive for high-throughput quantification, which often relies on inverse regression, like partial least squares regression. Due to a multivariate nature of spectroscopic measurements an analyte can be quantified in presence of interferences. However, if the model is not fully selective against interferences, analyte predictions may be biased. The degree of model selectivity against an interferent is defined by the inner relation between the regression vector and the pure interfering signal. If the regression vector is orthogonal to the signal, this inner relation equals zero and the model is fully selective. The degree of model selectivity largely depends on calibration data quality. Strong correlations may deteriorate calibration data resulting in poorly selective models. We show this using a fructose-maltose model system. Furthermore, we modify the NIPALS algorithm to improve model selectivity when calibration data are deteriorated. This modification is done by indified algorithm can be viewed as a fusion between ordinary least squares regression and partial least squares regression and may be very useful when knowledge of some (but not all) interfering species is available.The complementary role of hair in testing scenarios has expanded across the spectrum of toxicological and clinical monitoring investigations and, over the last 20 years, hair analysis has gained increasing attention and recognition. Moreover, a great deal of attention has been paid to the miniaturisation of extraction procedures, minimising/eliminating toxic organic solvents consumption, making them user-friendly and rapid, in addition to maximising extraction efficiency. The aim of this work is to provide a critical review of the advances observed over the last 5 years in the use of miniaturised approaches for sample clean-up and drug pre-concentration in hair analysis. There have been major improvements in some well-established microextraction approaches, such as liquid phase microextraction, mainly through the use of supramolecular and ionic liquids. In addition, new developments have also been reported in solid phase microextraction, driven by d-SPE applications. In the last 5 years, a total of 69 articles have been published using some type of microextraction technique for hair specimens, thus justifying the relevance of a critical review of innovations, improvements and trends related to these miniaturised approaches for sample preparation.