A low-cost, enzyme-coupled fluorescent assay for rapid quantification of glycolysis rate of cells.

Glycolysis is the predominant energy-yielding metabolic pathway in most cancer cells and rapidly proliferating cells. Currently available methods for glycolysis rate analysis are either time-consuming or cost-intensive/specialized equipment-dependent. The present study demonstrates a convenient, fast, and low-cost enzyme-coupled fluorometric assay for rapid quantification of glycolysis rate in small amount of cells. This assay involves the oxidation of cell-secreted lactate to produce hydrogen peroxide (H2O2) and subsequent conversion of Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) to fluorometric resorufin, in the presence of lactate oxidase (LOx) and peroxidase. High detection sensitivity and stability were realized by optimization of assay medium composition, enzyme and substrate concentration, and assay procedure. The lower limit of detection on HeLa cells was achieved on 50 cells per sample and the optimized linear range of the detection was 250-7000 cells per sample (r2 = 0.9842). The repetitive intraday and interday measurements of HeLa cell provided small variance and were highly agreeable with the results of endpoint method, which is a conventional validated method but detects lactate in relatively long time of larger cell population. The present assay was successfully applied on measuring the glycolytic parameters of human cancer cells (HeLa, HepG2) and mouse immune cells (T cells, macrophages), indicating great potential for wide application in cancerous and immunological research.

Low-cost calcium fluorometry for long-term nanoparticle studies in living cells.

Calcium fluorometry is critical to determine cell homeostasis or to reveal communication patterns in neuronal networks. Recently, characterizing calcium signalling in neurons related to interactions with nanomaterials has become of interest due to its therapeutic potential. However, imaging of neuronal cell activity under stable physiological conditions can be either very expensive or limited in its long-term capability. Here, we present a low-cost, portable imaging system for long-term, fast-scale calcium fluorometry in neurons. Using the imaging system, we revealed temperature-dependent changes in long-term calcium signalling in kidney cells and primary cortical neurons. Furthermore, we introduce fast-scale monitoring of synchronous calcium activity in neuronal cultures in response to nanomaterials. Through graph network analysis, we found that calcium dynamics in neurons are temperature-dependent when exposed to chitosan-coated nanoparticles. These results give new insights into nanomaterial-interaction in living cultures and tissues based on calcium fluorometry and graph network analysis.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

Development of rapid, sensitive and selective fluorimetric method for determination of 1-naphthalene acetic acid in cucumber by using magnetite-molecularly imprinted polymer.

In this study, a novel method based on the determination of 1-naphthalene acetic acid with the usage of magnetite-molecularly imprinted polymer prior to fluorimetric detection has been developed. Magnetite-molecularly imprinted polymer has been used for the first time as selective adsorbent for the determination of 1-naphthalene acetic acid. The adsorption capacity of the synthesized polymer was found to be 2.18 ±â€¯0.36 mg g-1 (n = 3). Limit of detection (LOD) and limit of quantification (LOQ) of the method were found to be 0.75 and 2.50 µg L-1, respectively. Linearity of the calibration graph for the proposed method was observed within the range of 20-700 µg L-1. The proposed method seems to be rapid where the detection procedure for 1-naphthalene acetic acid can be completed within a total time of 1 h. The same imprinted polymer can be used for the determination of 1-naphthalene acetic acid with quantitative sorption and recovery values repeatedly for at least ten times. The effects of some potential organic interferences were investigated. Proposed method has been successfully applied to determine 1-naphthalene acetic acid in cucumber, where the recoveries of the spiked samples were found to be in the range of 93.7-104.5%. Characterization of the synthesized polymer was also evaluated. By combining the high capacity, cheapness, reusability and selectivity of the magnetic adsorbent with the dynamic calibration range, rapidity, simplicity, and sensitivity of fluorimetry, the proposed method seems to be an ideal method for the determination of trace levels of 1-naphthalene acetic acid.

A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism.

In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine-Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine-Zn(II)-pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstract A simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine-Zn(II) complex.

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.

A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers.

BACKGROUND: Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup. METHODOLOGY: With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions. RESULTS/CONCLUSIONS: We show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators.

Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1.

Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.

A new fluorimetric method for the detection and quantification of siderophores using Calcein Blue, with potential as a bacterial detection tool.

The presence of microorganisms in biological fluids like urine and blood is an indication of vulnerability to infections. Iron is one of the important micronutrients required for bacterial growth. In an iron-deficit environment, bacteria release high-affinity iron-chelating compounds called siderophores which can be used as non-invasive target molecules for the detection of such pathogens. However, only limited reagents and procedures are available to detect the presence of these organic molecules. The present study aims at detecting the presence of siderophores in the iron-depleted media, exploiting the reversible quenching of Calcein Blue and iron(III) complex. The fluorescence of Calcein Blue is known to be quenched in the presence of iron(III); if a stronger chelator removes this ion from the fluorophore, the fluorescence of the fluorophore is regained. This behaviour of the fluorophore was exploited to detect and quantify siderophores down to 50 and 800 nM equivalent of standard siderophore, deferroxamine mesylate (desferal) in Dulbecco's PBS and siderophore quantification (SPQ) medium, respectively. The siderophores released by pathogens, equivalent to standard desferal, were in the range of 1.29 to 5.00 µM and those for non-pathogens were below 1.19 µM. The simple, sensitive and cost-effective method performed in a 96-well plate was able to detect and quantify iron chelators within 7-8 h of incubation.

Exclusion-Based Capture and Enumeration of CD4+ T Cells from Whole Blood for Low-Resource Settings.

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods.

Clinical applicability and prognostic significance of molecular response assessed by fluorescent-PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study.

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent-PCR (F-PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F-PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D-J, IGK V-J and KDE rearrangements. The applicability of F-PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non-molecular response; median progression-free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5-year survival: 75%) versus 66 months in the non-molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non-immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5-year survival: 95%) versus 69 months (P = 0·004), respectively. F-PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM.

A portable analyser for the measurement of ammonium in marine waters.

A portable ammonium analyser was developed and used to measure in situ ammonium in the marine environment. The analyser incorporates an improved LED photodiode-based fluorescence detector (LPFD). This system is more sensitive and considerably smaller than previous systems and incorporates a pre-filtering subsystem enabling measurements in turbid, sediment-laden waters. Over the typical range for ammonium in marine waters (0­10 mM), the response is linear (r(2) = 0.9930) with a limit of detection (S/N ratio > 3) of 10 nM. The working range for marine waters is 0.05­10 mM. Repeatability is 0.3% (n =10) at an ammonium level of 2 mM. Results from automated operation in 15 min cycles over 16 days had good overall precision (RSD = 3%, n = 660). The system was field tested at three shallow South Florida sites. Diurnal cycles and possibly a tidal influence were expressed in the concentration variability observed.

Simultaneous multiple target detection in real-time loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

Low-cost optical detectors and flow systems for protein determination.

Two miniature, fibreless, compact and highly integrated flow-through optoelectronic detectors dedicated for photometric and fluorimetric determination of proteins have been developed. Both detectors operating according to paired-emitter-detector-diode methodology are constructed only of light emitting diodes and therefore their total cost is extremely low. The photometric detector is dedicated for protein determination according to Bradford method based on detection of protein adduct with Coomassie Brilliant Blue. The fluorimetric detector allows determination of proteins after reaction with fluorescamine. Both developed detectors have been incorporated into economic flow systems constructed of microsolenoid valves and pumps. The resulting multicommutated/multipumping flow analysis systems enable detection of albumins and globulins at ppm levels, thus they are useful for protein determination in diluted samples of physiological fluids.

In vitro monitoring of natural thorium in urine using fluorimeter.

A relatively less expensive and less time consuming radio analytical technique for quantitative determination of Th(nat) in urine at mBq level is developed and reported in this paper. Th in urine is co-precipitated with Ca(3)(PO(4))(2) from wet oxidized urine matrix and the precipitate is dissolved in HNO(3) and evaporated to dryness. The residue is dissolved in 3M HCl and 200mg of Na-EDTA is added to mask Ca(2+), Mg(2+) and Fe(3+) ions. Th(4+) is extracted into 0.01M PC-88A (2-ethyl hexyl phosphonic acid mono-2-ethylhexyl ester), dissolved in toluene from the experimentally optimized pH 2.5 ± 0.3 in aqueous phase. Th(4+) is stripped into 8.0M HCl and evaporated to dryness. The content of the beaker is dissolved in pH 1.8 HCl and complexed with 3-hydroxy flavone. The sample is excited at 397 nm and fluorescence intensity is measured at 462 nm. The detailed study of the method is presented in this paper. Interference study on elements that are normally present in urine and other actinides (if present) is also given.

Development of a simple and low-cost device for fluorometric determination of selenium in water samples.

A portable spectrofluorometer device comprising an ultraviolet LED (380 nm) as a light source, an LED driver, a microsyringe as a cell, an optical fiber cable, a CCD spectrometer and a personal computer was used on-site. The device works on a battery for 3 h without the need to re-charge. The consumptions of reagents and sample solution can be reduced by using the device. Using fluorescein solution as a standard, the performance of this device was compared with that of a bench-top spectrofluorometer. The device applicability was demonstrated by the determination of selenium content in river water as a model of hazardous elements in the environment. Selenium reacted with 2,3-diaminonaphthalene to form piazselenol, which was then extracted with cyclohexane. The determination was carried out with both the portable device and the spectrofluorometer. The entire process was completed in approximately 15 min. The recovery of selenium in a river-water sample ranged from 104-112% and the detection limit was 0.5 microg L(-1).

Low-cost fluorimetric determination of radicals based on fluorogenic dimerization of the natural phenol sesamol.

A novel fluorimetric method for determining radicals using the natural phenol sesamol as a fluorogenic reagent is reported. In this assay, sesamol was reacted with aqueous radicals to yield one isomer of a sesamol dimer exclusively. The dimer emitted purple fluorescence near 400 nm around neutral pH, where it assumed the monoanionic form. This method was applied to the straightforward detection of radical nitric oxide (NO). The ready availability of sesamol should enable rapid implementation of applications utilizing this new assay, particularly in high-throughput analysis or screening.

Microfluorimeter with disposable polymer chip for detection of coeliac disease toxic gliadin.

Coeliac disease is an inflammatory disease of the upper small intestine and results from gluten ingestion in genetically susceptible individuals, and is the only life-long nutrient-induced enteropathy. The only treatment is a strict gluten-free diet and the longer the individual fails to adhere to this diet, the greater the chance of developing malnutrition and other complications. The existence of reliable gluten free food is crucial to the well-being of the population. Here we report on a microfluorimeter device for the in situ detection of gliadin in foodstuffs, which could be used for a rapid control of raw materials in food processing, as well as for process control of gliadin contamination. The microfluorimeter is based on a reflector that is used inside a microfluidic chip, exploiting various strategically placed reflective or totally metallised mirrors for efficient collection of the fluorescent light emitted in a large solid angle. The chip is capable of executing five assays in parallel and has been demonstrated to possess detection sensitivity applicable to fluoroimmunoassays. Various immunoassay formats exploiting fluorescence detection, using enzyme/fluorophore labels were developed and compared in terms of sensitivity, ease of assay, assay time and compatibility with buffer used to extract gliadin from raw and cooked foodstuffs, with the best performance observed with an indirect competition assay using a fluorophore-labelled anti-mouse antibody. This assay was exploited within the microfluorimeter device, and a very low detection limit of 4.1 ng/mL was obtained. The system was observed to be highly reproducible, with an RSD of 5.9%, for a concentration of 50 ng/mL of gliadin applied to each of the five channels of the microfluorimeter. Biofunctionalised disposable strips incorporated into the microfluorimeter were subjected to accelerated Arrhenius thermal stability studies and it was demonstrated that strips pre-coated with gliadin could be stored for approximately 2 years at 4 degrees C, with no discernable loss in sensitivity or detectability of the assay. Finally, the microfluorimeter was applied to the analysis of commercial gluten-free food samples, and an excellent correlation with routine ELISA measurements was obtained. The developed microfluorimeter should find widespread application for on-site execution of fluoroimmunoassays.

A luminescence lifetime assisted ratiometric fluorimeter for biological applications.

In general, the most difficult task in developing devices for fluorescence ratiometric sensing is the isolation of signals from overlapping emission wavelengths. Wavelength discrimination can be achieved by using monochromators or bandpass filters, which often lead to decreased signal intensities. The result is a device that is both complex and expensive. Here we present an alternative system--a low-cost standalone optical fluorimeter based on luminescence lifetime assisted ratiometric sensing (LARS). This paper describes the principle of this technique and the overall design of the sensor device. The most significant innovation of LARS is the ability to discriminate between two overlapping luminescence signals based on differences in their luminescence decay rates. Thus, minimal filtering is required and the two signals can be isolated despite significant overlap of luminescence spectra. The result is a device that is both simple and inexpensive. The electronic circuit employs the lock-in amplification technique for the signal processing and the system is controlled by an onboard microcontroller. In addition, the system is designed to communicate with external devices via Bluetooth.

Making medical devices accessible: a drug-resistant malaria detection system.

People living in areas of the world that are affected by disease, famine, or poverty could have their lives drastically improved by currently available electronic technologies, but the cost, complexity and/or limited operating environments of devices which employ these technologies can make it impossible or impractical for many of those in-need populations to actually acquire and make use of them. The barriers to acquisition and use are understandable. Most medical device companies are located in the wealthier countries, as are their clientele, and, to these companies, taking technological advances and either creating new products, or make their pre-existing products more powerful is the logical business and scientific progression. As such, devices seen in production that would be useful are often hospital grade with high-accuracy and varying amounts of adjustability so as to allow them to be used in performing a variety of functions. But it is exactly this accuracy and flexibility that makes them too expensive and complex to easily be acquired and used in environments that have limited financial and skill resources. In this paper, as an example of how to make presently inaccessible technology accessible, a method of detecting drug-resistant malaria strains in laboratories is analyzed, barriers to use of such a system in a poorer, malaria affected region are looked at, and a device is designed and a prototype built that is simple and affordable. Such a system not only allows collection of epidemiological information, but also empowers doctors and researchers to investigate new drugs without expensive laboratories.