Focusing ion funnel-assisted ambient electrospray enables high-density and uniform deposition of non-spherical gold nanoparticles for highly sensitive surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is a powerful technique for detecting trace amounts of analytes. However, the performance of SERS substrates depends on many variables including the enhancement factor, morphology, consistency, and interaction with target analytes. In this study, we investigated, for the first time, the use of electrospray deposition (ESD) combined with a novel ambient focusing DC ion funnel to deposit a high density of gold nanoparticles (AuNPs) to generate large-area, uniform substrates for highly sensitive SERS analysis. We found that the combination of ambient ion focusing with ESD facilitated high-density and intact deposition of non-spherical NPs. This also allowed us to take advantage of a polydisperse colloidal solution of AuNPs (consisting of nanospheres and nanorods), as confirmed by finite-difference time domain (FDTD) simulations. Our SERS substrate exhibited excellent capture capacity for model analyte molecules, namely 4-aminothiophenol (4-ATP) and Rhodamine 6G (R6G), with detection limits in the region of 10-11 M and a relative standard deviation of <6% over a large area (∼500 × 500 µm2). Additionally, we assessed the quantitative performance of our SERS substrate using the R6G probe molecule. The results demonstrated excellent linearity (R2 > 0.99) over a wide concentration range (10-4 M to 10-10 M) with a detection limit of 80 pM.

MCR-ALS with sample insertion constraint to enhance the sensitivity of surface-enhanced Raman scattering detection.

The multivariate curve resolution-alternative least squares (MCR-ALS) algorithm was modified with sample insertion constraint to deconvolute the overlapping peaks in SERS spectra. The developed method was evaluated by the spectral data simulated using a Gaussian distribution function to generate two independent peaks corresponding to a capping agent and an analyte. The spectra were generated with different overlapping levels and various intensity ratios of the analyte to the capping agent. By using MCR-ALS with the sample insertion constraint, the peak of the capping agent was completely excluded to obtain a calibration model of the analyte with R2 > 0.95 under all conditions. Furthermore, our developed method was later applied to a real SERS measurement to quantify carbofuran (analyte) using the azo-coupling reaction with p-ATP (capping agent) on silver nanoparticles as a SERS substrate. A calibration model of derivative carbofuran phenol was generated with R2 = 0.99 and LOD = 28.19 ppm. To assess the performance of the calibration model, the model was used to estimate the concentration of carbofuran in an external validation set. It was found that the RMSE of prediction was only 2.109 with a promising R2 = 0.97.

Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation.

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Distinguishing Enantiomers by Tip-Enhanced Raman Scattering: Chemically Modified Silver Tip with an Asymmetric Atomic Arrangement.

Discrimination between enantiomers is achieved by tip-enhanced Raman scattering (TERS) using a silver tip that is chemically modified by an achiral para-mercaptopyridine (pMPY) probe molecule. Differences in the relative intensities of the pMPY spectra were monitored for three pairs of enantiomers containing hydroxy (-OH) and/or amino (-NH2 ) groups. The N: or N+ -H functionality of the pMPY-modified tip participates in hydrogen-bond interactions with a particular molecular orientation of each chiral isomer. The asymmetric arrangement of silver atoms at the apex of the tip induces an asymmetric electric field, which causes the tip to become a chiral center. Differences in the charge-transfer (CT) states of the metal-achiral probe system in conjunction with the asymmetric electric field produce different enhancements in the Raman signals of the two enantiomers. The near-field effect of the asymmetric electric field, which depends on the number of analyte functional groups capable of hydrogen-bond formation, improves the degree of discrimination.

Online preconcentration and determination of chondroitin sulfate, dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis.

Capillary electrophoresis with large-volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and -16 kV. Exploiting large-volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.

Identification of Volatile Compounds and Selection of Discriminant Markers for Elephant Dung Coffee Using Static Headspace Gas Chromatography-Mass Spectrometry and Chemometrics.

Elephant dung coffee (Black Ivory Coffee) is a unique Thai coffee produced from Arabica coffee cherries consumed by Asian elephants and collected from their feces. In this work, elephant dung coffee and controls were analyzed using static headspace gas chromatography hyphenated with mass spectrometry (SHS GC-MS), and chemometric approaches were applied for multivariate analysis and the selection of marker compounds that are characteristic of the coffee. Seventy-eight volatile compounds belonging to 13 chemical classes were tentatively identified, including six alcohols, five aldehydes, one carboxylic acid, three esters, 17 furans, one furanone, 13 ketones, two oxazoles, four phenolic compounds, 14 pyrazines, one pyridine, eight pyrroles and three sulfur-containing compounds. Moreover, four potential discriminant markers of elephant dung coffee, including 3-methyl-1-butanol, 2-methyl-1-butanol, 2-furfurylfuran and 3-penten-2-one were established. The proposed method may be useful for elephant dung coffee authentication and quality control.

Diagnosis of early-stage esophageal cancer by Raman spectroscopy and chemometric techniques.

Esophageal cancer is a disease with high mortality. In order to improve the 5 year survival rate after cancer treatment, it is important to develop a method for early detection of the cancer and for therapy support. There is increasing evidence that Raman spectroscopy, in combination with chemometric analysis, is a powerful technique for discriminating pre-cancerous and cancerous biochemical changes. In the present study, we used Raman spectroscopy to examine early-stage (stages 0 and I) esophageal cancer samples ex vivo. Comparison between the Raman spectra of cancerous and normal samples using a t-test showed decreased concentrations of glycogen, collagen, and tryptophan in cancerous tissue. Partial least squares regression (PLSR) analysis and self-organization maps (SOMs) discriminated the datasets of cancerous and normal samples into two groups, but there was a relatively large overlap between them. Linear discriminant analysis (LDA) based on Raman bands found in the t-test was able to predict the tissue types with 81.0% sensitivity and 94.0% specificity.

3D SERS Imaging Using Chemically Synthesized Highly Symmetric Nanoporous Silver Microparticles.

3D surface-enhanced Raman scattering (SERS) imaging with highly symmetric 3D silver microparticles as a SERS substrate was developed. Although the synthesis method is purely chemical and does not involve lithography, the synthesized nanoporous silver microparticles possess a regular hexapod shape and octahedral symmetry. By using p-aminothiophenol (PATP) as a probe molecule, the 3D enhancement patterns of the particles were shown to be very regular and predictable, resembling the particle shape and exhibiting symmetry. An application to the detection of 3D inhomogeneity in a polymer blend, which relies on the predictable enhancement pattern of the substrate, is presented. 3D SERS imaging using the substrate also provides an improvement in spatial resolution along the Z axis, which is a challenge for Raman measurement in polymers, especially layered polymeric systems.

Selective colors reflection from stratified aragonite calcium carbonate plates of mollusk shells.

An interaction between the incident light and the structural architecture within the shell of Asian green mussel (Perna viridis) induces observable pearlescent colors. In this paper, we investigate the influence of the structural architecture on the expressed colors. After a removal of the organic binder, small flakes from crushed shells show vivid rainbow reflection under an optical microscope. An individual flake expresses vivid color under a bright-field illumination while become transparent under a dark-field illumination. The expressed colors of the aragonite flakes are directly associated with its structural architecture. The flakes with aragonite thickness of 256, 310, and 353 nm, respectively, appear blue, green, and red under an optical microscope. The spectral simulation corroborates the experimentally observed optical effects as the flakes with thicker aragonite layers selectively reflected color with longer wavelengths. Flakes with multiple aragonite thicknesses expressed multi-color as the upper aragonite layers allow reflected colors from the lower layers to be observed.

Poly(N-isopropylacrylamide)-stabilized gold nanoparticles in combination with tricationic branched phenylene-ethynylene fluorophore for protein identification.

Gold nanoparticles stabilized by thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM-AuNPs) were prepared by surface grafting of thiol-terminated PNIPAM onto citrate-stabilized AuNPs. The color change of the PNIPAM-AuNPs solution from red to blue-purple without precipitation when the solution was heated to 40 °C, above the lower critical solution temperature (LCST) of PNIPAM, indicated the thermoresponsive property of the synthesized AuNPs. PNIPAM-AuNPs were used to detect proteins by chemical nose approach based on fluorescence quenching of fluorophore by AuNPs. An array-based sensing platform for detection of six proteins, namely bovine serum albumin, lysozyme, fibrinogen, concanavalin A, hemoglobin, holo-transferrin human can be successfully developed from the PNIPAM-AuNPs having different molecular weights (4 and 8 kDa) and conformation (varied heat treatment from 25 to 40 °C) in combination with a tricationic branched phenylene-ethynylene fluorophore. From principal component analysis (PCA) followed by linear discriminant analysis (LDA), 100% accuracy of protein classification using a leave-one-out (LOO) approach can be achieved by using only two types of PNIPAM-AuNPs.

Chemometric analysis of spectroscopic data on shape evolution of silver nanoparticles induced by hydrogen peroxide.

The study on the shape evolution of metal nanoparticles (MNPs) is crucial to gain an understanding on controlling the shape and size of metal nanostructures. In this work, a detailed study on shape evolution of silver (Ag) nanospheres to nanoplates induced by hydrogen peroxide (H2O2) was performed. According to the growth mechanism of Ag nanoplates, the spectrophotometric method combined with chemometric analysis has potential to reveal the structural evolution process as observed by surface plasmon resonance phenomena. The extinction spectra of the evolving nanostructures were analyzed by factor analysis and error indicator functions. Five major components attributed to the different particle shapes and sizes were theoretically predicted. Furthermore, the concentration profiles and pure spectra of these components were resolved using multivariate curve resolution-alternative least squares (MCR-ALS) analysis. The evolution profiles show that the spherical Ag particles systematically evolved into plate structures of different sizes. Larger nanoplates were obtained when higher concentrations of H2O2 were employed. An evidence of nanoplate disintegration was observed when a large amount of H2O2 was employed. The predicted structural morphologies of each component given by chemometric calculation were in excellent agreement with those observed by transmission electron microscope (TEM) images.

Paper spray mass spectrometry as an effective tool for differentiating coffees based on their geographical origins.

With the rising trend of valuing flavor complexity of coffees, means to distinguish the properties of individual coffee sources is vital to the sustainable growth of the coffee industry. Herein, paper spray mass spectrometry (PS-MS), a simple technique with little sample preparation, was used to collect mass data from aqueous extracts of coffees from various sources. Thereafter, principal component analysis and linear discriminant analysis were used to successfully classify coffee samples (with 80-100 % accuracy) from various studies including the differentiations of Arabica and Robusta coffees, Arabica coffees from different countries, Robusta coffees from different geographical locations, and Arabica coffees from different locations within the same province in Thailand. With further insight from significant test via Fisher weight determination, this method was proved to be practical for differentiating coffees based on types and geographical origins, thus paving the way for broader applications.

Rapid geographical indication of peppercorn seeds using corona discharge mass spectrometry.

With increasing demands for more rapid and practical analyses, various techniques of ambient ionization mass spectrometry have gained significant interest due to the speed of analysis and abundance of information provided. Herein, an ambient ionization technique that utilizes corona discharge was applied, for the first time, to analyze and categorize whole seeds of black and white peppers from different origins. This setup requires no solvent application nor gas flow, thus resulting in a very simple and rapid analysis that can be applied directly to the sample without any prior workup or preparation. Combined with robust data pre-processing and subsequent chemometric analyses, this analytical method was capable of indicating the geographical origin of each pepper source with up to 98% accuracies in all sub-studies. The simplicity and speed of this approach open up the exciting opportunity for onsite analysis without the need for a highly trained operator. Furthermore, this methodology can be applied to a variety of spices and herbs, whose geographical indication or similar intellectual properties are economically important, hence it is capable of creating tremendous impact in the food and agricultural industries.

Supervised self organizing maps for classification and determination of potentially discriminatory variables: illustrated by application to nuclear magnetic resonance metabolomic profiling.

The article describes the extension of the self organizing maps discrimination index (SOMDI) for cases where there are more than two classes and more than one factor that may influence the group of samples by using supervised SOMs to determine which variables and how many are responsible for the different types of separation. The methods are illustrated by an application in the area of metabolic profiling, consisting of a nuclear magnetic resonance (NMR) data set of 96 samples of human saliva, which is characterized by three factors, namely, whether the sample has been treated or not, 16 donors, and 3 sampling days, differing for each donor. The sampling days can be considered a null factor as they should have no significant influence on the metabolic profile. Methods for supervised SOMs involve including a classifier for organizing the map, and we report a method for optimizing this by using an additional weight that determines the relative importance of the classifier relative to the overall experimental data set in order to avoid overfitting. Supervised SOMs can be obtained for each of the three factors, and we develop a multiclass SOM discrimination index (SOMDI) to determine which variables (or regions of the NMR spectra) are considered significant for each of the three potential factors. By dividing the data iteratively into training and test sets 100 times, we define variables as significant for a given factor if they have a positive SOMDI in the training set for the factor and class of interest over all iterations.

Mouse urinary biomarkers provide signatures of maturation, diet, stress level, and diurnal rhythm.

Body fluids such as urine potentially contain a wealth of information pertaining to age, sex, social and reproductive status, physiologic state, and genotype of the donor. To explore whether urine could encode information regarding environment, physiology, and development, we compared the volatile compositions of mouse urine using solid-phase microextraction and gas chromatography-mass spectrometry (SPME-GC/MS). Specifically, we identified volatile organic compounds (VOCs) in individual urine samples taken from inbred C57BL/6J-H-2(b) mice under several experimental conditions-maturation state, diet, stress, and diurnal rhythms, designed to mimic natural variations. Approximately 1000 peaks (i.e., variables) were identified per comparison and of these many were identified as potential differential biomarkers. Consistent with previous findings, we found groups of compounds that vary significantly and consistently rather than a single unique compound to provide a robust signature. We identified over 49 new predictive compounds, in addition to identifying several published compounds, for maturation state, diet, stress, and time-of-day. We found a considerable degree of overlap in the chemicals identified as (potential) biomarkers for each comparison. Chemometric methods indicate that the strong group-related patterns in VOCs provide sufficient information to identify several parameters of natural variations in this strain of mice including their maturation state, stress level, and diet.

Variable selection using iterative reformulation of training set models for discrimination of samples: application to gas chromatography/mass spectrometry of mouse urinary metabolites.

The paper discusses variable selection as used in large metabolomic studies, exemplified by mouse urinary gas chromatography of 441 mice in three experiments to detect the influence of age, diet, and stress on their chemosignal. Partial least squares discriminant analysis (PLS-DA) was applied to obtain class models, using a procedure of 20,000 iterations including the bootstrap for model optimization and random splits into test and training sets for validation. Variables are selected using PLS regression coefficients on the training set using an optimized number of components obtained from the bootstrap. The variables are ranked in order of significance, and the overall optimal variables are selected as those that appear as highly significant over 100 different test and training set splits. Cost/benefit analysis of performing the model on a reduced number of variables is also illustrated. This paper provides a strategy for properly validated methods for determining which variables are most significant for discriminating between two groups in large metabolomic data sets avoiding the common pitfall of overfitting if variables are selected on a combined training and test set and also taking into account that different variables may be selected each time the samples are split into training and test sets using iterative procedures.

Rapid Discovery and Structure-Property Relationships of Metal-Ion Fluorescent Sensors via Macroarray Synthesis.

A macroarray immobilisation of fluorophores on filter papers for sensing metal ions by in-situ reductive amination and carbodiimide coupling is reported herein. Chemometric approaches resulted in a rapid discovery of sensors that can synergistically discriminate up to 12 metal ions with great prediction accuracies. Covalently bound on paper, sensoring scaffolds that were synthesised from the macroarray format can readily be adopted as practical paper-based sensors with great reusability and sensitivity, achieving the limit of detection at low nanomolar level with some repeating spotting. Lastly, the discovered scaffolds were also confirmed to be functional as unbound molecules, thus paving the way for more diverse applications.

Paper-based chemical reaction arrays as an effective tool for geographical indication of turmerics.

Geographical indications have gained increasing importance as a powerful marketing tool for highly valuable products especially foods. In this study, a unique and synergistic combination of chemical reaction arrays on paper and chemometric analysis was used to uncover geographical indication of turmerics, an important food ingredient in several cultures. The key to effective differentiation was based on the subtle differences in the compositions of compounds found in each sample, mainly curcumin and derivatives. When these compounds reacted with various reagents in the form of paper arrays, different optical and fluorescence profiles were generated, which can then be exploited by chemometrics. As a result, our strategy could provide up to 94% prediction accuracy without the need for any sophisticated instruments.

Cellular responses of periodontal ligament stem cells to a novel synthesized form of calcium hydrogen phosphate with a hydroxyapatite-like surface for periodontal tissue engineering.

Calcium hydrogen phosphate with a hydroxyapatite-like surface (CHP-HA) is a novel synthesized compound designed to overcome the limitations of bioactive ceramics. It was originally applied as nano-sized HA strips covering core plates to enhance the degree of interfacial attachment. The objective of the present study was to examine the cellular attachment, proliferation, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) on a CHP-HA substrate in comparison with conventional nanohydroxyapatite (NanoHA). The PDLSCs were cultivated with either CHP-HA or NanoHA for cellular attachment, proliferation, and osteogenic differentiation assay. Osteogenic differentiation was examined using quantitative polymerase chain reaction and immunofluorescence after confirmation by Alizarin red staining. We found that between 14 and 21 days, CHP-HA exhibited a well-organized matrix distribution, a high degree of cell proliferation, and a high level of Alizarin red staining in comparison to NanoHA. Expression of all the osteogenic markers examined was increased significantly relative to NanoHA at 14 days, but no significant differences in some osteogenic genes were found at 21 days. Immunofluorescence revealed stronger staining in the CHP-HA group. In conclusion, PDLSCs cultivated with this novel CHP-HA show enhanced cellular responses. We propose that CHP-HA may be a promising alternative biomaterial for periodontal tissue engineering.

High purity silver microcrystals recovered from silver wastes by eco-friendly process using hydrogen peroxide.

A simple, rapid, and environmentally friendly process using hydrogen peroxide, was developed for recovering high purity silver directly from industry and laboratory wastes. Silver ammine complex, [Ag(NH3)2]+Cl-, derived from AgCl were generated and then directly reduced using H2O2 to reliably turn into high purity microcrystalline silver (99.99%) examined by EDS and XRD. Morphology of the recovered silver microcrystals could be selectively tuned by an addition of poly(vinyl pyrrolidone). The main parameters in the recovering process including pH, concentration of Ag+ and the mole ratio of H2O2:Ag+ were carefully optimized though the central composite design (CCD). The optimized condition was employed for a trial recovery of 50 L silver ammine complex prepared from a collection of silver-wastes during 3-year research on industrial nanoparticle production. The recovered silver microcrystals >700 g could be recovered with 91.27%. The remaining solution after filtering of the recovered silver microcrystals can be used repeatedly (at least 8 cycles) without losing recovery efficiency. Matrix interferences including Pb2+ and Cl- play a minimal role in our silver recovery process. Furthermore, the direct usage of the recovered silver microcrystals was demonstrated by using as a raw material of silver clay for creating a set of wearable silver jewelries.