Analytical Methods (v.9, #43)

Front cover (6085-6086).

Contents list (6087-6091).

pH-Sensing fluorescence oligonucleotide probes based on an i-motif scaffold: a review by Anna Dembska; Patrycja Bielecka; Bernard Juskowiak (6092-6106).
Fluorescence-based approaches have found widespread applications in cell biology. pH-Sensing using fluorescent i-motifs is a potentially useful strategy for real-time, in vivo monitoring of important cellular processes. i-Motif scaffolds can serve as the recognition element for H+ and are highly programmable, which leads to probes that exhibit different dynamic pH ranges and possess slightly different transition midpoints. Moreover, various methodologies can be employed to obtain fluorescence signals, such as incorporation of fluorescent cytosine analogues and excimers or FRET labeling. An alternative label-free strategy for i-motif formation and as a consequence, for pH monitoring, is also presented within this review. Strategies used for the internalization of i-motif sensors are cited as well as an example of anchoring them to a cell membrane for extracellular measurements is described. In this review, we have focused on recent developments in pH-sensing fluorescence oligonucleotide probes based on an i-motif scaffold, especially in the context of intracellular sensing and bioimaging.

Green analytical flow method for the determination of total sulfite in wine using membraneless gas–liquid separation with contactless conductivity detection by Nattapong Chantipmanee; Waleed Alahmad; Thitaporn Sonsa-ard; Kanchana Uraisin; Nuanlaor Ratanawimarnwong; Thitirat Mantim; Duangjai Nacapricha (6107-6116).
A green analytical flow method was developed for the determination of total sulfite in white wine. The method employs the membraneless vaporization (MBL-VP) technique for gas–sample separation allowing direct analysis of wine. Sulfite in an aliquot of sample was converted to SO2 gas via acidification. Dissolution of the gas into the water acceptor led to a change in the conductivity of the acceptor which was monitored using a ‘capacitively coupled contactless conductivity detector’ (C4D) flow cell. Only a minute amount of common acid (100 μL of 1.5 mol L−1 H2SO4) is used. The MBL-VP unit was incorporated into the flow system to separate the SO2 gas from the wine sample using the headspace above the donor and acceptor compartments as a virtual membrane. The method provides a linear working range (10–200 mg L−1 sulfite) which is suitable for most wines with calibration equation y = (0.056 ± 0.002)x + (1.10 ± 0.22) and r2 = 0.998. Sample throughput is 26 samples h−1. The lower limit of quantitation (LLOQ = 3SD of blank per slope) is 0.3 mg L−1 sulfite for 20 s diffusion time with good precision (%RSD = 0.8 for 100 mg L−1 sulfite, n = 10). We also present a simple modification of the MBL-VP unit by the addition of a third cone-shaped reservoir to provide two acceptor zones leading to improvement in sensitivity of more than three-fold without use of heating to enhance the rate of diffusion of SO2.

Molecularly imprinted polymer-coated paper as a substrate for highly sensitive analysis using paper spray mass spectrometry: quantification of metabolites in urine by Thais P. P. Mendes; Igor Pereira; Marcella Rodrigues Ferreira; Andréa Rodrigues Chaves; Boniek Gontijo Vaz (6117-6123).
Here, we proposed an analytical approach based on the use of a molecularly imprinted polymer-coated paper substrate (MIP-CPS) for paper spray ionization mass spectrometry (PS-MS) to improve its specificity. The new substrate developed was applied to detect and quantify dopamine, sarcosine, and butyric acid in synthetic human urine without derivatization or complex sample pre-treatments. The urinary levels of these metabolites can be correlated with several pathological conditions including heart disease, stress, neurological disorders, cancerous tumors, and AIDS. Calibration curves exhibited R2 > 0.99 for dopamine, sarcosine, and butyric acid. LODs and LOQs were found at μg L−1 (parts-per-billion) for dopamine and sarcosine, and pg L−1 (parts-per-trillion) for butyric acid. Precision and accuracy showed coefficients of variation and relative errors less than 18% for almost all analyses. Recovery test results ranged between 95.5 and 117.7%. Finally, we compared the analytical performance of the MIP-CPS with a traditional paper substrate and ESI. The MIP-CPS showed a superior performance in detecting dopamine and avoiding the ionization suppression commonly observed during the analysis of complex biological samples such as urine.

Current techniques to analyze insulin, although efficient and accurate, usually require advanced scientific knowledge, chemical labeling, expensive equipment and/or are destructive. Ultrasonic spectroscopy is a viable alternative since it is simple to use, does not involve any sample preparation, and is non-destructive. Microfluidic ultrasonic spectroscopy detects particles through measuring the amount of acoustic energy transmitted at different frequencies. We have successfully performed acoustic spectroscopy to analyze insulin on the micro-scale, where the main advantage is that only 50 μL (vs. 1 mL) is required for sample analysis. To prove the efficacy of our device, we first measured fresh Sanofi U400 Insuman insulin over a range of frequencies. We then artificially aged insulin for six hours to compare the acoustic signature before and after protein aging. Next, to probe the dynamic range and sensitivity of our new method, we chose three frequencies and measured the progressive deterioration of insulin as it aged over time. Finally, we validated the performance of MIDAS against three benchmark tests: HPLC, ThT fluorescence assay and DLS. Specifically, we show that certain frequencies produce different spectra within 1 hour of accelerated aging. MIDAS compares well with benchmark tests of HPLC, ThT fluorescence assay and DLS. Such a device can be a complementary tool to help determine the stability of insulin in a cost-effective manner.

Separation and detection of free d- and l-amino acids in tea by off-line two-dimensional liquid chromatography by Xiangyu Wang; Huihui Wu; Rongying Luo; Donghai Xia; Zhengjin Jiang; Hai Han (6131-6138).
d-amino acids are currently paid attention to as new physiologically active substances. Foodstuffs and beverages containing d-amino acids are a matter of interest. Until now, the profiles of d-amino acids have not been reported in natural and fermented teas except theanine. In this study, an off-line 2D-HPLC method combining a Gemini C18 column and a CHIRALPAK® IC-3 column or a self-prepared poly(MQD-co-HEMA-co-EDMA) monolithic capillary column was employed for the separation and detection of d-amino acids in tea samples with the co-existence of a large amount of l-amino acids. The free amino acid fractions in longjing, black, oolong, and pu-erh tea samples were separated and collected after pre-column derivatization using 9-fluorenylmethoxycarbonyl (FMOC) chloride in the reversed-phase mode, and then were concentrated and separated to d- and l-forms on a chiral column. Among them, the d-form of isoleucine (Ile) (1.0–1.6%), alanine (Ala) (0.1–0.8%), phenylalanine (Phe) (0.1–0.4%), valine (Val) (0.2–0.3%), serine (Ser) (0.1–0.2%), aspartic acid (Asp) (0.2%), and proline (Pro) (0.1%) were detected in longjing and oolong teas. Differences between natural tea and fermented tea in the profiles of d-amino acids as well as the total amino acids were also observed. The results provided useful information for the bio-function research of d-amino acids in tea.

Detection of organophosphorus pesticides (OPPs) is crucial for food safety and environmental protection. Here, we propose an enzyme-free strategy for the detection of phosphorothiolate pesticides, a major class of OPPs with P–S bonds, by using hyaluronan–tyrosine–gold nanoparticles (HA–Tyr–AuNPs) as the colorimetric probe. The strategy is based on the concept that the surface of AuNPs can be directly modified by the hydrolyzate of the phosphorothiolate in a strongly basic HA–Tyr–AuNP colloidal solution, which in turn affects the aggregation state of AuNPs and hence produces a red-to-blue color change. This method is very simple in design and easy to operate. The detection requires only the addition of the analyte into the HA–Tyr–AuNP colloidal solution at room temperature. By virtue of the absorbance variations of the analytical system, the limit of detection for phorate is as low as 0.005 μg mL−1 (i.e., 0.006 mg kg−1), which is lower than the maximum residue limit (MRL) allowed by the European Union. More importantly, phorate residues in real samples can be directly detected by this method.

The potential of hyperspectral imaging in the spectral range of 1000–2500 nm with multivariate analysis for the prediction of oil and protein concentrations in five peanut cultivars was investigated. Quantitative partial least squares regression (PLSR) models were established using the extracted spectral data from hyperspectral images and the reference measured oil and protein concentrations. The PLSR models established using the whole spectral data pretreated by the multiplicative scatter correction (MSC) method showed good results for predicting the oil concentration with a determination coefficient (RP2) of 0.945 and root mean square errors of prediction (RMSEP) of 0.196 and for predicting the protein concentration with RP2 of 0.901 and RMSEP of 0.441. In addition, eight optimal wavelengths were selected for protein and oil contents, respectively, using the regression coefficients of the PLSR analysis and used for simplifying the obtained models. The simplified PLSR models also showed good performances with Rp2 of 0.933 and 0.912 for predicting oil and protein concentrations. The whole results demonstrated that the NIR hyperspectral imaging technique coupled with chemometric analysis is a promising tool for rapid and non-destructive determination of oil and protein concentrations in peanut kernels and has the potential to develop a multispectral imaging system for future on-line detection of peanut quality.

An effective signal enhancement strategy for sensing cysteine based on aluminium-initiated emission by Wu Zou; Xuejiao Chen; Cheng Huan; Tingting Gu; Jiaoyun Xia; Can Chen; Fuchun Gong (6155-6160).
An efficient signal enhancement strategy for the detection of cysteine (Cys) was developed, which is based on the combination of a Cys-reduced reaction and the chelation of Al3+ with the Cys-promoted product. The Cys probe S-DPM was prepared by adding a 2,4-dinitrobenzenesulfonyl group onto a 2′-hydroxyldihydropyrimidobenzimidazole derivative (DPM). Upon treatment with Cys, S-DPM was reduced and the hydroxyl group was released in the product DPM, thereby enabling an excited-state intramolecular proton transfer (ESIPT) process. When Al3+ ions were added, the fluorescence signal was dramatically increased owing to the transformation of the DPM into DPM-Al3+ complexes with a very high fluorescence quantum yield (0.865). The proposed method allows the analysis of Cys by modulating the switch of the fluorescence of S-DPM with Cys ranging from 0.01 to 4.5 μM. The detection limit is 17 nM. As expected, the S-DPM probe was successfully applied for the determination of Cys in human serum and plasma samples with recoveries ranging from 97.2.0% to 105.5%. Fluorescence imaging experiments showed that S-DPM can track Cys in living cells. ESIPT compounds are frequently used for the construction of Cys probes, but this method measures the fluorescence of only a part of the Cys-reduced products. The use of S-DPM coupled with Al3+ overcomes this drawback.

SmartIHC-Analyzer: smartphone assisted microscopic image analytics for automated Ki-67 quantification in breast cancer evaluation by Suman Tewary; Indu Arun; Rosina Ahmed; Sanjoy Chatterjee; Chandan Chakraborty (6161-6170).
As with other cancers, cell proliferation is one of the indicative hallmarks of breast cancer evaluation. The expression of human Ki-67, being a nuclear protein, has strong association with the proliferation of cancer cells. The proliferation index from Ki-67 is evaluated by immunohistochemical (IHC) study by discriminating positively (brown color) and negatively (blue color) stained nuclei of cells through manual counting. In practice, this evaluation process is highly dependent on an expert-pathologist, as well as being time consuming and prone to inter-observer variability. To circumvent current challenges in IHC image analysis, we introduce SmartIHC-Analyzer, an efficient smartphone assisted microscopy for automatic scoring of Ki-67 protein expression. A universal 3D printed adapter is fabricated to attach a smartphone to the eye-piece of the conventional microscope to acquire microscopic images of stained tissue slides. IHC image acquisition, analytics, visualization and automated reporting for Ki-67 have been developed and integrated in an android smartphone for easy use in any pathological set-up. Color deconvolution followed by morphological top-hat transformation and segmentation is performed to extract stained cells for automated scoring. The proposed SmartIHC-Analyzer app was tested on 30 cases of Ki-67 stained tissue samples and compared with the score given by expert pathologists with variability in illumination conditions. The results had high similarity with manual scoring by the expert pathologists (Pearson's correlation coefficient r = 0.97) and the average absolute variation from pathologists' score was evaluated as 6.21%; which is highly significant. The results have been compared with available state-of-the art ImmunoRatio software where the results are found to be promising. It can be highlighted that SmartIHC-Analyzer can be used in point-of-care diagnostics for instant automatic and augmented reporting of Ki-67 protein expression. The APK file of the SmartIHC-Analyzer is free to use and can be obtained by emailing the corresponding author.

Evaluation of sialic acid based on electrochemical cytosensor with 3D micro/nanostructured sensing interface by Shangshang Ma; Sisheng Hu; Qi Wang; Yuhong Liu; Guangyao Zhao; Qicheng Zhang; Chun Mao; Bo Zhao (6171-6176).
Sialic acid (SA), a glycoprotein associated with many pernicious diseases, has been applied to quantify cancer cells. In this paper, a novel electrochemical cytosensor with three-dimensional (3D) micro/nanostructured sensing interface, which can provide a better platform for cell adhesion, was utilized to detect the expression of SA from the cell surface. The hollow horn-like PPy (hPPy) film and chitosan–Au nanoparticles (CS–Au NPs) were electrodeposited on stainless steel and then, combined with a targeting lectin molecule of Sambucus nigra agglutinin (SNA), for sensing A549 human lung cancer cells based on the molecular recognition between SNA and SA. The morphologies, wettability and cytotoxicity of CS–Au/hPPy were investigated by scanning electron microscope (SEM), energy dispersive spectrum (EDS), water contact angle test and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays. Furthermore, the electrochemical performances of this cytosensor with a 3D micro/nanostructured CS–Au/hPPy sensing interface were investigated. Under optimal conditions, the proposed cytosensor exhibited a good linear relationship, wide linear range of the cell concentration from 10 to 1.0 × 107 cells per mL and a detection limit as low as 2 cells per mL (S/N = 3). Moreover, the cytosensor also had good stability and specificity to analyze the over-expressed SA on living cells, implying that the new sensing interface we proposed may have a huge potential application in the study of tumor cells and greatly promote the cancer diagnosis and treatment in early stages.

A robust and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of four endogenous hormones and exogenous contaminants including progesterone, bisphenol A (BPA), 4-nonylphenol (4-NP) and chloramphenicol (CAP) in infant formula powdered milk. After adding isotope internal standards, the samples were first extracted with ethyl acetate–cyclohexane and then extracted by a dissolution–salting out-ethyl acetate extraction method (anhydrous sodium sulfate was used as the salt) for enhanced extraction of target analytes. Subsequently, the extraction solution was concentrated and purified by using a HLB solid phase extraction (SPE) cartridge (150 mg/6 mL, 30 μm, Waters Co., USA). Four target analytes were separated on a Phenomenex kinetex C18 column (100 mm × 3.0 mm, 2.6 μm) by gradient elution with methanol and water as mobile phases, detected by MS/MS in multiple reaction monitoring (MRM) mode and quantified by the internal standard method with progesterone-d9, BPA-d4, CAP-d5, and 4-n-NP-d4 as the isotope internal standards. The total time for the detection was 7 min. The developed method was validated in terms of sensitivity, linearity and range, recovery, matrix effect and precision. The results indicated that four target analytes displayed excellent linearity in their corresponding concentration ranges, with favorable correlation coefficients greater than 0.99. The limits of quantitation (LOQs) for these target analytes were in the range of 0.1–2.5 μg kg−1. The mean recoveries were calculated at three concentration levels spiked in negative infant formula powdered milk samples, and the values were found between 81.9% and 103% with relative standard deviation (RSD) values ranging from 2.9% to 7.0% for intra-day precision (n = 6) and ranging from 5.8% to 8.0% for inter-day precision (n = 5). The validated method was successfully applied to determine the concentrations of target analytes in real samples, and progesterone and 4-NP were respectively detected in 73 and 24 samples with concentrations in the range of 1.5–90.5 μg kg−1 and 13.5–36.8 μg kg−1, respectively. Fortunately, BPA and CAP were detected in none of the tested samples.

Highly sensitive and label-free determination of thiram residue using surface-enhanced Raman spectroscopy (SERS) coupled with paper-based microfluidics by Jiaji Zhu; Quansheng Chen; Felix Y. H. Kutsanedzie; Mingxiu Yang; Qin Ouyang; Hui Jiang (6186-6193).
In this study, a paper-based microfluidic surface-enhanced Raman spectroscopy (SERS) device was employed for the determination of trace level thiram. The paper-based microfluidic device was fabricated by cutting a hydrophilic region which had been printed on the filter paper and then pasting it onto sellotape. The Au@Ag nanoparticles (NPs) were synthesized with a 30 nm Au core and 7 nm Ag shell and used as the SERS probe. The synthesized nanoparticles were dropped in one of the sample adding zones of the paper-based microfluidics and the thiram solution was dropped in another one. The solutions flowed through their own channels by capillary action and mixed together in the reaction chamber. The optimization studies on the use of paper-based microfluidic devices are discussed. In SERS measurements, the intensity of the peak at 1143 cm−1 was highly sensitive, and so it was chosen as an ideal peak for the quantitative analysis of the concentration of thiram solution. The limit of detection (LOD) of thiram was as low as 1.0 × 10−9 mol L−1, and the relative standard deviation (RSD) results analyzed at 10 random spots in the SERS measurement area were all below 10%. The recovery values of thiram in adulterated tea samples were from 95% to 110%. All these results suggest that this proposed method is a prospective candidate for trace level thiram detection.

The degree of substitution (DS) of highly acetylated cellulose acetate (CA) was quantitatively evaluated by FTIR. Seven standard samples, the DS ranging from 1.80 to 2.85, prepared by mixing appropriate amounts of commercial cellulose triacetate and commercial cellulose were analyzed for their DS by a FTIR method, and the relationship between the DS and the ratios of specific peak absorbance intensity or integral area calculated from FTIR spectra was confirmed to be a polynomial fitting of the second degree. The DS values of CA prepared by heterogeneous acetylation under different conditions determined by FTIR based on the resulting quadratic polynomial equations were compared with those determined by a titration method. The results showed that the DS value was well predicted by FTIR spectra and the average percentage errors were lower than 2.31%. The specific peak parameter ratio can effectively evaluate and monitor the acetylation process. Additionally, the results of dynamic analysis indicated that the heterogeneous acetylation reaction followed a second-order kinetic model when the DS value exceeded 2.0.

The widely used nitroblue tetrazolium (NBT) colorimetric method for superoxide anion radical (O2˙) detection suffers from limitations such as formazan solubility and interference from similar electron donors. This work reports the development of a novel assay for superoxides, where O2˙ generated by a NADH/PMS/O2 system oxidizes the N,N-dimethyl-p-phenylene diamine (DMPD) reagent to pink-colored DMPD-semiquinone (DMPDQ) cationic radicals retained by a cation exchange Nafion® membrane and measured colorimetrically. Antioxidants exhibiting superoxide radical scavenging activity (SRSA) cause less DMPDQ radical production, resulting in an attenuated color intensity on the Nafion® membrane, the absorbance difference (ΔA) at 514 nm being proportional to the antioxidant concentration. The developed method was applied to 14 antioxidant compounds including trolox, phenolic and hydroxycinnamic acids, flavonoids, and thiols, and the linear concentration ranges, calibration equations (as ΔA versus concentration), and trolox-equivalent antioxidant capacities of these antioxidants were established. The results were discussed in the light of structure–activity relationships. This method was used for measuring the SRSA of antioxidant mixtures and a green tea extract. The Nafion® membrane-contacted green tea extract was transferred to an online-HPLC post-column CUPRAC system to measure unconsumed catechins. The percentage inhibitions for green tea found with the developed method were compared to those measured by the reference NBT method.

Back cover (6213-6214).