A ratiometric fluorescence probe for bisulfite detection in live cells and meat samples.

The development of reliable probe technology for the detection of bisulfite (HSO3-) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO3- in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO3- triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO3- internalization and production endogenously. The capability of probe EHDI for HSO3- was then validated by traditional HPLC technology, enabling accurately detect HSO3- in beef samples. The successful development of this probe thus offers a new tool for investigating HSO3- in situ in food and biological conditions.

Facile fluorescence detection of malachite green in fish using molecularly imprinted polymers doped CdTe quantum dots based system.

Malachite green (MG) possesses high toxicity, therefore, the detection of MG in fish tissues is of vital importance. A novel core-shell MIPs doped CdTe quantum dots coated silica nanoparticles (CdTe-MIP/SiO2 NPs) were synthesized via a simple one-pot strategy. The materials were characterized carefully. The resulting CdTe-MIP/SiO2 NPs were coated on the thin layer chromatography plate, and coupled with miniaturized fluorimeter for fluorescence detection of MG in fish samples. The resulting CdTe-MIP/SiO2 NPs based system possessed good linearity (0.01 âˆ¼ 20 µmol/L), high recoveries (98.36 %∼101.45 %) and low detection limit (3.7 nmol/L) for MG. Furthermore, CdTe-MIP/SiO2 NPs based system were employed to measure fish samples spiked with MG, meanwhile, HPLC was utilized to evaluate the accuracy and reliability. And the paired t-test was conducted to evaluate differences between fluorescence method and HPLC, P > 0.05 means no significant difference was observed, the results demonstrated that both fluorescence method and HPLC are suitable for MG analysis.

A Simple and Low-cost Preliminary Quantification of Target Membrane Protein in Single Cells.

To study the heterogeneity of target membrane proteins in single cells with cellular integrity, we proposed a simple and low-cost method to obtain the copy number of the membrane proteins. HeLa cells were labeled by FITC affinity bodies specifically targeting HER2 membrane proteins. The immunolabeled HeLa cells were quantified by a laboratory-built laser induced fluorescence detector. A series of fluorescent microspheres with known number of FITC molecules on the surface were used to establish the calibration curve, instead of the standard fluorescent solutions, because the morphology of the microspheres was similar to the cells, and the distribution of FITC on the spheres were similar to the distribution of HER2 on the HeLa. The fluorescence intensity of the cells was converted to the molecule number of HER2 by the calibration curve. A capillary electrophoresis system was used to drive the microspheres and cells through the detection window. The copy number of HER2 in HeLa cells ranged from 4,036 to 1,224,920 ± 100 (2.5-97.5%), and the median of copy numbers were 104,438 ± 100 per cell. This method for measuring low-abundance membrane proteins can be utilized for the initial exploration of proteomics in ordinary laboratories.

A 96-well plate UV fluorometer based on micro fluorescence detector array and dynamic zero correction algorithm.

A 96-well plate UV fluorometer was developed and evaluated. Eight micro fluorescence detectors close to each other were used as detector array for 8 channels. Each detector employed an UV light emitting diode (LED) as light source and a photodiode (PD) with an amplifier circuit as optoelectronic detector. The optical paths of the detectors were designed by ray tracing method to avoid crosstalk between wells. Simultaneously scanning and detecting of 8 channels saves scanning time and improves detection efficiency. The scanning time of the 96-well plate was about 80 s. A dynamic zero correction algorithm was proposed to solve the problem of measurement accuracy reduction caused by the background fluorescence differences between plates and wells under irradiation of UV light. The measurement repeatability (RSD) for 1 µg/L 7-Diethylamino-4-methylcoumarin sample was 2.25%. Compared with the fixed zero correction method, the limit of detection (LOD), measurement repeatability, and average relative error were improved by 3.3, 2.7, and 4.5 times, respectively. The proposed method is robust and can be applied to different analysis systems. The developed fluorometer has great potential in high-throughput rapid detection of food safety and life sciences.

Fabrication and application of molecularly imprinted polymer doped carbon dots coated silica stationary phase.

Facing the difficulties in chromatographic separation of polar compounds, this investigation devotes to developing novel stationary phase. Molecularly imprinted polymers (MIPs) have aroused wide attention, owing to their outstanding selectivity, high stability, and low cost. In this work, a novel stationary phase based on carbon dots (CDs), MIP layer, and silica beads was synthesized to exploit high selectivity of MIPs, excellent physicochemical property of CDs, and outstanding chromatographic performances of silica microspheres simultaneously. The MIP doped CDs coated silica (MIP-CDs/SiO2) stationary phase was systematically characterized by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET) surface area measurement, and carbon elemental analysis. Furthermore, the chromatographic performance of the MIP-CDs/SiO2 column was thoroughly assessed by using a wide variety of compounds (including nucleosides, sulfonamides, benzoic acids, and some other antibiotics). Meanwhile, the separation efficiency of the MIP-CDs/SiO2 stationary phase was superior to other kinds of stationary phases (e.g. nonimprinted NIP-CDs/SiO2, MIP/SiO2, and C18-SiO2). The results demonstrated that MIP-CDs/SiO2 column exhibited best performance in terms of chromatographic separation. For all tested compounds, the resolution value was not less than 1.60, and the column efficiency of MIP-CDs/SiO2 for thymidine was 22,740 plates/m. The results further indicate that the MIP-CDs/SiO2 column can combine the good properties of MIP, CDs, with those of silica microbeads. Therefore, the developed MIP-CDs/SiO2 stationary phase can be applied in the separation science and chromatography-based techniques.

A miniaturized hydrogen flame ionization detector based on integrated nozzle assembly and embedded sealing structure.

A modified miniaturized hydrogen flame ionization detector (m-FID) was developed and evaluated. An integrated nozzle assembly was constructed to solve the gas leakage caused by adhesive crack during repeated high-low temperature processes or vibration. An embedded sealing structure was designed to realize the face sealing, thus improving the sealing stability and reliability of the m-FID. Polyimide was employed as seal and insulation material to ensure the detector can be used at 300 °C for a long term. The hydrogen and air consumption of the m-FID was 12 mL/min and 110 mL/min, which is about 1/3 of the FID gas consumption of commercial laboratory instruments. The limit of detection (LOD) for n-hexadecane was 3.2 × 10-12 g/s, with a linear response range of nearly 5 orders of magnitude. Finally, it was installed onto an on-site gas chromatograph to detect drug samples with wide boiling point range from room temperature up to 535 °C.

Peltier thermoelectric cooler improves both the signal-to-noise ratio and warm-up time of high-power LED induced fluorescence detector and application to aflatoxins.

High-power LED induced fluorescence detector (HP-LED-IF) suffers from problems of large noise and poor baseline stability. In this study, a Peltier thermoelectric cooler (PTC) was utilized to stabilize the HP-LED junction temperature of a HP-LED-IF to reduce the baseline noise for the first time. Compared with traditional fan cooling, the signal-to-noise ratio was improved to 3.8 times, and the warm-up time was shortened by 64.4%. For application, a 365/450 nm HP-LED-IF was constructed and coupled with HPLC for detection of aflatoxins. The limits of detections (LODs, 3 times peak-to-peak noise) for aflatoxin G2 and B2 were 1.2 and 1.0 pg/mL, respectively. In-situ photochemical derivatization reaction of G1 and B1 in 28 µL detection cell within 2.1 s flow time was found surprisingly for the first time, which enhanced the fluorescence signal by about 10 times. The LODs for aflatoxin G1 and B1 were 3.4 and 2.4 pg/mL, respectively. These LODs are among the lowest values that have been reported. This study provides a key technique to improve both the signal-to-noise ratio and warm-up time of HP-LED-IFs and a novel in-situ derivatization method for aflatoxins G1 and B1.

Enhancement of Chemiluminescence Intensity of S2* in Non-premixed Hydrogen Microjet Flame in the Photometric Detector for Sulfur Detection.

A transparent quartz rod (q) placed vertically on top of a non-premixed hydrogen microjet flame in a flame photometric detector (qFPD) was developed and evaluated for sulfur detection. The microjet flame burned around the quartz rod because of Coanda effect, forming an extended downstream flame zone with a relatively low temperature between 550 and 650 °C, which is favorable to the formation of S2*. The emission intensity of S2* and the signal-to-noise ratio (SNR) of sulfur response were enhanced 2.6- and 2.1-fold, respectively. It was found that the quartz rod of diameter 4 mm with a tip shape of semicircle placed 6 mm above the nozzle yielded the highest SNR. The limits of detection (LOD) for seven kinds of tested sulfur-containing compounds of qFPD were 0.3-0.5 pg S s-1, which is 5-7 times better than that of commercially available FPD detectors (LOD: 1.6-2.8 pg S s-1). The selectivity of sulfur over carbon was 105 on qFPD when the SNR for the mass flow rate of S and C atoms was ∼3 times. It was the first time that a quartz rod was used vertically on top of a microjet hydrogen-rich flame in FPD to enhance the chemiluminescence of S2* and improve the LOD down to 0.3-0.5 pg S s-1.

Spherical Dichroic Reflector Improves Limit of Detection in Laser-Induced Fluorescence Detection.

A miniature laser-induced fluorescence (mLIF) detector utilizing a novel spherical dichroic reflector (SDR), an unconventional long working distance high magnification objective, an uncommon broadband emission-matched excitation filter pair, and a silicon-based photodiode detector assembly instead of a photomultiplier tube was developed and evaluated. The detection cell was placed at the spherical center of the SDR instead of the regular focus, yielding a 1.8× signal-to-noise ratio (SNR) improvement. Different from previous works, the use of a 40× objective with a long working distance of 5.38 mm and a broadband BP 527-70 nm emission filter with matched BP 450-30 nm excitation filter improved SNR to 4.6× and 1.9×, respectively. By flow injection analysis (FIA) evaluation, the limit of detection (LOD; 3σ method) for fluorescein sodium was 1.5 × 10-13 M or 8.9 fluorescein molecules in 98 pL detection volume, which was the lowest level of LIFs evaluated by FIA mode. The analysis of three kinds of amino acids with LODs at sub pM to fM level (the lowest levels, hundreds of times lower than previous works using normal capillary) demonstrated the potential of the mLIF in ultratrace analysis of biological and environmental samples, including low copy molecules in a single cell.

A miniaturized and high sensitive dual channel fluorimeter based on compact collinear optical arrangement.

A miniaturized and high sensitive dual channel fluorimeter was developed and evaluated. It employed collinear optical arrangement, a 365 nm and a 470 nm light emitting diodes (LEDs) as light sources, two photodiodes (PDs) integrated with pre-amplifiers as optoelectronic detectors, and a 12.5 mm × 12.5 mm × 45 mm (width × length × height) quartz cuvette as detection cell. The optical parameters such as spectrum compatibility of dual channel, reshaping lens, the common optical path length (COPL), the common focus lens (CFL), as well as working distance of the cuvette were optimized carefully. It was found that the use of shortened optical path and common focal lens could improve the sensitivity of the dual channel fluorimeter significantly. The limits of detection (LODs) for coumarin, aflatoxin B1, fluorescein sodium, and vitamin B2 were 0.002 µg L-1, 0.006 µg L-1, 0.008 µg L-1, and 0.03 µg L-1, respectively. The dual channel fluorimeter can be used for detection of several categories of substance, such as mycotoxins, polycyclic aromatic hydrocarbons, fluorescein, vitamins, and pathogenic microorganisms etc. As a key component, it can also find application in different disciplines such as fluorescent PCR instruments and 96-well plate fluorescence analyzer.

Aqueous extraction followed by dispersive solid phase extraction with in situ derivatization for the determination of aflatoxins in traditional Chinese medicines.

A green sample preparation method based on aqueous extraction followed by dispersive solid phase extraction (d-SPE) with in situ derivatization (ISD) was developed for the determination of aflatoxins (AFs) in traditional Chinese medicines (TCMs). AFs in TCMs were extracted by alkaline aqueous solution and converted to substituted coumaric acids. Then, mixed-mode anion exchange (MAX) sorbent was used to isolate and enrich the substituted coumaric acids. During the elution by acetonitrile/trifluoroacetic acid solution, AFs were reconstructed and in situ derivatized. Several parameters affecting the procedure were evaluated. The developed preparation method coupled with high performance liquid chromatography-fluorescence detection was successfully applied for AFs determination in TCMs. The limit of detection (LOD) reached 10 pg/mL for AFs. Good linearity was obtained in three orders of magnitude with correlation coefficients ranging from 0.9996 to 0.9999. The relative recoveries of the method were between 72.7% and 114.5% with intra- and inter-day relative standard deviations (RSDs) less than 9.5% and 10.1%, respectively. The method was successfully applied to determine AFs in 15 kinds of TCMs in China, with the results verified by IAC standard method.

A flame photometric detector with a silicon photodiode assembly for sulfur detection.

A flame photometric detector with a silicon photodiode assembly instead of a photomultiplier tube for sulfur detection was developed and evaluated. The photosensitive area of photodiode, the optical design, and band-pass filters, were optimized. It was found that the optimal photosensitive area of the photodiode was 100 (mm)2, and three focus lenses combined with a broad band-pass filter of 378/52 nm and a QB21 glass yielded the best result. This design fully utilized the wide emission spectrum of S2*, the response characteristics of silicon photodiode, and effective absorption of strong emission spectrums of OH* at wavelength around 310 nm by QB21 glass. The limits of detection for nine kinds of sulfur containing compounds were between 5.8 × 10-12 to 9.5 × 10-12 g s-1. This mode provided a linear response of 3 orders of magnitude for compounds being tested and a selectivity of sulfur over carbon of 105. It is demonstrated for the first time that the overall performance of the flame photometric detector integrated with a silicon photodiode assembly work at room temperature was comparable to a conventional detector coupled with a photomultiplier tube, with advantages of short equilibration time, robust to electromagnetic interference and vibration, and low cost. The new detector can find wide application in gas chromatography and on-line monitoring instruments for sulfur measurement.

Quantification of Low Copy Number Proteins in Single Cells.

We have developed an ultrasensitive and highly selective method to quantify low copy number intracellular proteins in a single cell using a low-cost laser-induced fluorescence (LIF) detector and a BV605 fluorescent probe. Active caspase3 proteins in cells were labeled by corresponding antibody-BV605 fluorescent binding, and a cell was injected into a 20 cm × 50 µm i.d. capillary column, followed by in situ lysis and capillary electrophoresis (CE)-LIF analysis. About seven active caspase3 protein molecules in a detection volume of 91 pL could be detected. In our method, cross-bounding proteins other than active caspase3 could be separated and distinguished by differences of retention time. By using Si photodiode assembly as a fluorescent detector instead of PMT, the dynamic range of the LIF is over 4 orders of magnitude. In this experiment, we found that the number of active caspase3 molecules in 98 single Jurkat cells were from 629 to 12171, reflecting significant heterogeneity among the cells although they were from the same batch. For extended application, it could also be applied to quantify other types of low copy number proteins in a single cell as long as the corresponding antibodies are provided. This high-sensitive method could also be a promising tool for earlier cancer diagnosis and related disease pathway research which is relevant to low copy number proteins. In addition, this low-cost system could also be easily expanded to an array system for high-throughput quantitation of low copy proteins in single cells.

One step rapid dispersive liquid-liquid micro-extraction with in-situ derivatization for determination of aflatoxins in vegetable oils based on high performance liquid chromatography fluorescence detection.

A rapid dispersive liquid-liquid micro-extraction (DLLME) with in-situ derivatization method for extraction and purification of aflatoxins (AFs) in vegetable oils was developed and evaluated. Oil extract, dichloromethane and trifluoroacetic acid were mixed and injected into water to form a cloudy solution. AFs in the oil were extracted into the numerous liquid droplets (with diameters from a few microns to dozens of microns) of extractant, where derivatization was carried out in situ. The proposed sample preparation method was coupled with high performance liquid chromatography with fluorescence detection (HPLC-FLD) for determination of four AFs in vegetable oils. The method showed excellent linearity in three orders of magnitude, good relative recoveries, good repeatability and high sensitivity with limits of detection in range of 0.005-0.03 ng/mL. The accuracy of the method was also verified by certified reference sample. Finally, different kinds of vegetable oils from the local supermarket were analyzed.

A novel HPLC flow cell integrated UV light emitting diode induced fluorescence detector as alternative for sensitive determination of aflatoxins.

A novel UV light emitting diode induced fluorescence detector (LED-IF) with HPLC flow cell was designed and evaluated for determination of aflatoxins. Ray tracing method was employed to optimize the design of the optical components (such as reshaping thin lens for light source and fluorescence collection), and optical structure, and the geometry of flow cell. An ordinary UV LED with wavelength of 370 nm and radiant power of 5 mW was used as excitation light source. A photoelectric amplifier AccuOpt2000 with light sensitivity of 10-5∼10-4lx was utilized for fluorescence detection, instead of a photo multiplier tube (PMT). The lowest detection limit (LOD) of the LED-IF was 0.077 ppb for aflatoxin B1 by HPLC method without derivatization. The LED-IF was then coupled with a homemade iodine derivatization device, which was connected to the HPLC column outlet for determination of aflatoxins in edible oil and peanut samples. It was found that the edible oil sample contained aflatoxin B1 and B2 at average level of 7.14 (n = 3) and 0.30 ng/g (n = 3), respectively; while the peanut sample contained aflatoxin B1 and B2 at average level of 9.22 (n = 3) and 1.36 ng/g (n = 3), respectively. The overall power consumption of the LED-IF was 1 W/12 V. The sensitivity of the LED-IF was similar to commercial fluorescence detectors, which utilized a pulse Xe lamp as excitation light source and a PMT for detection, with power consumption of 150 W.

A compact and low-cost laser induced fluorescence detector with silicon based photodetector assembly for capillary flow systems.

A compact and low-cost laser induced fluorescence (LIF) detector based on confocal structure for capillary flow systems was developed and applied for analysis of Her2 protein on single Hela cells. A low-power and low-cost 450 nm laser diode (LD) instead of a high quality laser was used as excitation light source. A compact optical design together with shortened optical path length improved the optical efficiency and detection sensitivity. A superior silicon based photodetector assembly was used for fluorescence detection instead of a photomultiplier (PMT). The limit of detection (LOD) for fluorescein sodium was 3 × 10-12 M or 165 fluorescein molecules in detection volume measured on a homemade capillary electroosmotic driven (EOD)-LIF system, which was similar to commercial LIFs. Compared to commercial LIFs, the whole volume of our LIF was reduced to 1/2-1/3, and the cost was less than 1/3 of them.

A facile and high sensitive micro fluorimeter based on light emitting diode and photodiode.

A facile and high sensitive micro fluorimeter was developed and evaluated. It employed light emitting diode (LED) as light source, cuvette as detection cell, and photodiode (PD) as optoelectronic detector. Optical and electronic parameters were optimized and demonstrated. A high power LED was chosen, which could irradiate the inner area of the cuvette completely at the same time with divergence angle as small as possible. The optimum LED brought 2.5 times signal-to-noise ratio (SNR) enhancement. Using reflector at the opposite direction of excitation light path doubled SNR. The amplifier circuit of PD was deeply investigated to achieve high sensitivity, low noise, and good stability. The limit of detection (LOD) of fluorescein isothiocyanate (FITC) and chlorophyll at SNR = 3 were 10pM ~ 0.004 ppb and 0.05 ppb, respectively. Basing on the principle structure, a portable fluorimeter for fungimycin detection was developed using a low power UV LED as light source. The LOD for aflatoxin B1 was 0.1 ppb.

Signal-to-noise ratio enhancement of the compact light-emitting diode-induced fluorescence detector.

To enhance the SNR (signal-to-noise ratio) of the compact light-emitting diode-induced fluorescence detector (LED-FD) ([14]), key parameters including LED and its coupling style, pinhole diameter, calibration, and refractive index matching fluid (RIMF) were optimized. A 20 mA LED with light intensity of 8400 mcd was used as new light source. To optimize pinhole diameter, a theoretical analysis based on a three-dimensional (3D) view of the detection area was proposed and validated experimentally. A calibration principle from the 3D perspective was proposed. The detection flow cell and the collection fiber were adjusted to be coplanar. RIMF (glycerol) was applied to enhance SNR. The performance of the improved LED-FD was evaluated by flow injection analysis (FIA). The limit of detection (LOD) was determined as 0.15 nM sodium fluorescein (SNR=3). Compared with our previous work, a five-fold enhancement on the SNR was obtained. The LOD was about five times higher than that of commercial Agilent G1321A FLD which employed a xenon pulse lamp.

Hollow fiber-based liquid-liquid-liquid micro-extraction with osmosis: I. Theoretical simulation and verification.

Osmosis in hollow fiber-based liquid-liquid-liquid micro-extraction (HF-LLLME) was validated and utilized to improve enrichment factor of extraction in this study. When donor phase (sample solution) with higher ion strength than acceptor phase (extraction phase) was used, osmosis was established from acceptor phase, through organic membrane to donor phase. The mass flux expression of analytes across the organic membrane was established based on the convective-diffusive kinetic model, and the kinetic process for HF-LLLME with osmosis was simulated. Simulation results indicated that osmosis from acceptor phase to donor phase can increase enrichment factor of HF-LLLME, accelerate extraction process, and even result in the distribution ratio of analytes between donor and acceptor phase exceeding their partition coefficient. This phenomenon was verified by the experimental data of extraction with six organic acids and four organic bases as the model analytes.

A compact and highly sensitive light-emitting diode-induced fluorescence detector for capillary flow systems.

A compact and highly sensitive light-emitting diode-induced fluorescence detector (LED-FD) was constructed and evaluated. The LED excitation beam was restricted by a 200 µm diameter pinhole on a 55 µm thick lighttight film. The LED, excitation filter, pinhole, and capillary flow cell were placed tightly against each other without either optical lens or excitation fiber. The performance of the LED-FD was evaluated by flow injection analysis (FIA) of sodium fluorescein solution. The limit of detection (LOD) was determined as 0.75 nM (SNR=3). Compared with our previous work, a 3.5-fold enhancement on the signal-to-noise ratio (SNR) was obtained. The system exhibited linear responses in the concentration range of 2-200 nM (R=0.9993). The error of repeatability was within 2.0% R.S.D. on peak height in a ten-repeated injection of 5 nM solution. The reproducibility between devices was within 2.2% R.S.D. on peak height of 10nM fluorescein solution. Finally, the LED-FD was compared with commercial fluorescence detector, and similar fluorescence signal intensity was obtained.