Low-cost (<€5), open-source, potential alternative to commercial spectrophotometers.

Spectrophotometry is a fundamental technique in many areas of science, with many applications and uses. The cost of spectrophotometers has acted as a barrier on the teaching and use of the technique. Here, we provide open-source plans to a 3D-printed cuvette holder with an interchangeable narrow-spectral bandwidth light-emitting diode (LED) block that can be used in conjunction with a smartphone's ambient light sensor (ALS) to perform spectrophotometry. A Lego version with an interchangeable LED block is also presented. Results from the smartphone spectrophotometer in comparison with commercially available spectrophotometers demonstrated functionality, and the model may have many applications, especially in indirect spectrophotometry, such as in the protein assay shown here. The plans for the 3D-printed model are freely available on GitHub, as are editable files to allow customisation by users. We would encourage users to share adaptations with the scientific community.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.

The cost-effectiveness of a novel SIAscopic diagnostic aid for the management of pigmented skin lesions in primary care: a decision-analytic model.

OBJECTIVES: Pigmented skin lesions are commonly presented in primary care. Appropriate diagnosis and management is challenging because the vast majority are benign. The MoleMate system is a handheld SIAscopy scanner integrated with a primary care diagnostic algorithm aimed at improving the management of pigmented skin lesions in primary care. METHODS: This decision-model-based economic evaluation draws on the results of a randomized controlled trial of the MoleMate system versus best practice (ISRCTN79932379) to estimate the expected long-term cost and health gain of diagnosis with the MoleMate system versus best practice in an English primary care setting. The model combines trial results with data from the wider literature to inform long-term prognosis, health state utilities, and cost. RESULTS: Results are reported as mean and incremental cost and quality-adjusted life-years (QALYs) gained, incremental cost-effectiveness ratio with probabilistic sensitivity analysis, and value of information analysis. Over a lifetime horizon, the MoleMate system is expected to cost an extra £18 over best practice alone, and yield an extra 0.01 QALYs per patient examined. The incremental cost-effectiveness ratio is £1,896 per QALY gained, with a 66.1% probability of being below £30,000 per QALY gained. The expected value of perfect information is £43.1 million. CONCLUSIONS: Given typical thresholds in the United Kingdom (£20,000-£30,000 per QALY), the MoleMate system may be cost-effective compared with best practice diagnosis alone in a primary care setting. However, there is considerable decision uncertainty, driven particularly by the sensitivity and specificity of MoleMate versus best practice, and the risk of disease progression in undiagnosed melanoma; future research should focus on reducing uncertainty in these parameters.

A quick, convenient and economical method for the reliable determination of methylglyoxal in millimolar concentrations: the N-acetyl-L-cysteine assay.

The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The "gold standard" method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-L-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-L-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents.

Determination of oxybutynin in pharmaceuticals via reaction with mixed acids anhydrides: application to content uniformity testing.

Sensitive and simple spectrophotometric (Method I) and spectrofluorimetric (Method II) methods were developed and validated for the determination of oxybutynin HCl (OXB) in its dosage forms. The method was based on the reaction of OXB with malonic acid anhydride in acetic acid anhydride to form a highly yellow colored product that was measured at 375 nm spectrophotometrically. The same reaction product exihibits strong fluorescence that was measured at 440 nm after excitation at 390 nm. The factors affecting formation and stability of the reaction product were carefully studied and optimized, and the reaction mechanism was postulated. The absorbance-concentration plot is rectilinear over the range 4-40 µg/mL with LOD of 1.12 µg/mL and LOQ of 3.39 µg/mL. The fluorescence-concentration plot is rectilinear over the range 0.5-6 µg/mL with LOD of 0.11 µg/mL and LOQ of 0.33 µg/mL. The method was applied to the analysis of commercial tablets Detronin® and Uripan®. Statistical comparison of the results with those of the reference method revealed good agreement and proved that there were no significant difference in the accuracy and precision between the two methods respectively. The study was extended to content uniformity testing.

Chemometrics-assisted spectrophotometric method for simultaneous determination of vitamins in complex mixtures.

An advanced independent component analysis algorithm (MILCA) is applied for simultaneous chemometric determination of fat- and water-soluble vitamins in complex mixtures. The analysis is based on the decomposition of spectra of multicomponent mixtures in the UV region. The key features of the proposed method are simplicity, accuracy, and reliability. Comparisons between the new algorithm and other established methods (MCR-ALS, SIMPLISMA, other ICA techniques) were made. Our results indicate that in most cases, MILCA is comparable or even outperforms other chemometrics methods taken for comparisons. The influence of different factors (abundance of components, noise, step of spectral scan, and scan speed) on decomposition performance has been investigated. The optimal conditions for spectroscopic registration have been identified. The proposed method was used for analysis of model mixtures and real objects (multivitamin drugs, food additives, and energy drinks). The resolved concentrations match well with the declared amounts and the results of reference methods.

Determination of naltrexone and 6beta-naltrexol in human blood: comparison of high-performance liquid chromatography with spectrophotometric and tandem-mass-spectrometric detection.

We present data for a comparison of a liquid-chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) and a high-performance liquid-chromatographic method with column switching and UV spectrophotometric detection. The two methods were developed for determination of naltrexone and 6beta-naltrexol in blood serum or plasma aiming to be used for therapeutic drug monitoring to guide the treatment of patients with naltrexone. For the high-performance liquid chromatography (HPLC)/UV detection, online sample cleanup was conducted on Perfect Bond C(18) material with 2% (vol/vol) acetonitrile in deionized water. Drugs were separated on a C(18) column using 11.5% (vol/vol) acetonitrile and 0.4% (vol/vol) N,N,N,N-tetramethylethylenediamine within 20 min. LC-MS/MS used naltrexone-d (3) and 6beta-naltrexol-d (4) as internal standards. After protein precipitation, the chromatographic separation was performed on a C(18) column by applying a methanol gradient (5-100%, vol/vol) with 0.1% formic acid over 9.5 min. The HPLC/UV method was found to be linear for concentrations ranging from 2 to 100 ng/ml, with a regression correlation coefficient of r (2) > 0.998 for naltrexone and 6beta-naltrexol. The limit of quantification was 2 ng/ml for naltrexone and 6beta-naltrexol. For the LC-MS/MS method the calibration curves were linear (r(2) > 0.999) from 0.5 to 200 ng/ml for both substances, and the limit of quantification was 0.5 ng/ml. The concentrations measured by the two methods correlated significantly for both substances (r(2) > 0.967; p < 0.001). Both methods could be used for therapeutic drug monitoring. The HPLC/UV method was advantageous regarding automatization and costs, whereas LC-MS/MS was superior with regard to sensitivity.

Automated determination of uranium(VI) at ultra trace levels exploiting flow techniques and spectrophotometric detection using a liquid waveguide capillary cell.

Rapid and fully automated multisyringe flow-injection analysis (MSFIA) with a multi-pumping flow system (MPFS) coupled to a long path-length liquid waveguide capillary cell (LWCC) is proposed for the determination of uranium(VI) at ultra trace levels. On-line separation and pre-concentration of uranium is carried out by means of a TRU resin. After elution, uranium(VI) is spectrophotometrically detected after reaction with arsenazo-III. Combination of the MSFIA and MPFS techniques with the TRU-resin enables the analysis to be performed in a short time, using large sample volumes and achieving high selectivity and sensitivity levels. A detection limit of 12.6 ng L(-1) (ppt) is reached for a 100-mL sample volume. The versatility of the proposed method also enables pre-concentration of variable sample volumes, enabling application of the analysis to a wide concentration range. Reproducibility of better than 5% and a resin durability of 40 injections should be emphasized. The developed method was successfully applied to different types of environmental sample matrices with recoveries between 95 and 108%.

Automated measurement of urinary creatinine by multichannel kinetic spectrophotometry.

Urinary creatinine analysis is required for clinical diagnosis, especially for evaluation of renal function. Creatinine adjustment is also widely used to estimate 24-h excretion from spot samples. Few convenient validated approaches are available for in-house creatinine measurement for small- to medium-scale studies. Here we apply the Jáffe reaction to creatinine determination with zone fluidic multichannel kinetic spectrophotometry. Diluted urine sample and reagent, alkaline picric acid, were mixed by a computer-programmed dispenser and rapidly delivered to a four-channel detection cell. The absorbance change was monitored by a flow-through light-emitting diode-photodiode-based detector. Validation results against high-performance liquid chromatography-ultraviolet (HPLC-UV)/mass spectrometry (MS) are presented. Responses for 10-fold diluted samples were linear within clinically relevant ranges (0-250 mg/L after dilution). The system can analyze 70 samples per hour with a limit of detection of 0.76 mg/L. The relative standard deviation was 1.29% at 100 mg/L creatinine (n=225). Correlation with the HPLC (UV quantitation/MS confirmation) system was excellent (linear, r2=0.9906). The developed system allows rapid, simple, cost-effective, and robust creatinine analysis and is suitable for the analysis of large numbers of urine samples.

A simple and cost-effective method, as an appropriate alternative for visible spectrophotometry: development of a dopamine biosensor.

In this study, a new, simple, fast and inexpensive method as an alternative to visible spectrophotometry is developed. In this method the cells containing the sample solution were scanned with a scanner, then the color of each cell was analyzed with software written in visual basic (VB 6) media to red, green and blue values. The cells were built by creating holes in the Plexiglas sheet. The dimensions of identical cells were examined by Cr (III) solution with known concentrations. The validity of this new method was studied by determination of dopamine (DA) without using any other reagent. The parameters which affect the system were optimized. The comparison between the current and traditional UV-Vis spectrophotometry methods was studied and the results revealed similar trends in both methods. The developed method was successfully applied to the determination of dopamine in serum and urine without using any pretreatment. Finally comparing the results obtained in the developed method showed that microwave irradiation of the solution can decrease the experimental time, increase sensitivity and improve the limit of detection.

Simple and sensitive colorimetric detection of cysteine based on ssDNA-stabilized gold nanoparticles.

In this paper, we demonstrate a simple and sensitive colorimetric detection of cysteine based on the cysteine-mediated color change of ssDNA-stabilized gold nanoparticles (AuNPs). Cysteine is capable of absorbing onto AuNPs surfaces via the strong interaction between its thiol group and gold. ssDNA molecules which stabilize AuNPs against salt-induced aggregation are removed away by cysteine encapsulation on the AuNPs surfaces, resulting in a characteristic color change of AuNPs from red to blue as soon as salt is added. The ratio of absorptions at 640 to 525 nm (A(640)/A(525)) is linear dependent on the cysteine concentration in the range from 0.1 to 5 microM. Furthermore, amino acids other than cysteine cannot mediate the color change under the identical conditions due to the absence of thiol groups, suggesting the selectivity of the proposed method toward cysteine. The employment of complicated protocols and sophisticated processes such as the preparation of modified AuNPs are successfully avoided in design to realize the simple and low-cost cysteine detection; and the high sensitivity and low cost of the method is favorable for practical applications.

Spectrophotometric determination of urea in dermatologic formulations and cosmetics.

A rapid, relatively sensitive, and low-cost method for the determination of water-soluble urea content in dermatological therapy products and cosmetics is proposed using a new spectrophotometric assay with water as the only extraction solvent. Spectrophotometric methods involve addition of a known excess of bromate to urea in an acid medium, followed by the determination of residual bromine and chlorine reacting with methyl orange and measurement of absorbance at 505 nm. The absorbance increases linearly with urea concentration (r = 0.9998). The systems obey Beer's law for 6 - 90 microg ml(-1). The calculated apparent molar absorbance values are found to be 4.537 x 10(3) dm(3) mol(-1) cm(-1) and the Sandell's sensitivity is 0.013 microg cm(-2). The variables affecting the rate of the reaction were investigated. The relative standard deviation for five-replication determination of 60 microg ml(-1) urea was 2.1% and the detection limit of the method is 0.34 ng ml(-1).

An ultra-low-cost smartphone octochannel spectrometer for mobile health diagnostics.

With the rapid development and proliferation of mobile devices with powerful computing power and the ability of integrating sensors into mobile devices, the potential impact of mobile health (mHealth) diagnostics on the public health is drawing researchers' attention. We developed a Smartphone Octo-channel Spectrometer (SOS) as a mHealth diagnostic tool. The SOS has nanoscale wavelength resolution, is self-illuminated from the smartphone itself, and is ultra-low cost (less than $20). A user interface controls the optical sensing parameters and precise alignment. After calibrating and testing the SOS by quantifying protein concentrations, we clinically validated the SOS by comparing the diagnostic performance of our device with that of a clinical spectrophotometer. About 180 serum samples from de-identified patients with 4 types of autoantibodies were blindly read the ELISA results. The accuracy of the SOS achieved 100% across the clinical reportable range compared with the FDA-approved instrument. Furthermore, the self-illuminated SOS only requires about half of the light intensity of the FDA-approved instrument to achieve clinical-level sensitivity. The low-energy-consumption and low-cost SOS enables point-of-care spectrophotometric sensing in low-resource areas, and can be integrated into point-of-care diagnostic systems for rapid multiplex readout and analysis at patient bedside or at home.

Smartphone based optical spectrometer for diffusive reflectance spectroscopic measurement of hemoglobin.

We report a miniature, visible to near infrared G-Fresnel spectrometer that contains a complete spectrograph system, including the detection hardware and connects with a smartphone through a microUSB port for operational control. The smartphone spectrometer is able to achieve a resolution of ~5 nm in a wavelength range from 400 nm to 1000 nm. We further developed a diffuse reflectance spectroscopy system using the smartphone spectrometer and demonstrated the capability of hemoglobin measurement. Proof of concept studies of tissue phantoms yielded a mean error of 9.2% on hemoglobin concentration measurement, comparable to that obtained with a commercial benchtop spectrometer. The smartphone G-Fresnel spectrometer and the diffuse reflectance spectroscopy system can potentially enable new point-of-care opportunities, such as cancer screening.

A multichannel smartphone optical biosensor for high-throughput point-of-care diagnostics.

Current reported smartphone spectrometers are only used to monitor or measure one sample at a time. For the first time, we demonstrate a multichannel smartphone spectrometer (MSS) as an optical biosensor that can simultaneously optical sense multiple samples. In this work, we developed a novel method to achieve the multichannel optical spectral sensing with nanometer resolution on a smartphone. A 3D printed cradle held the smartphone integrated with optical components. This optical sensor performed accurate and reliable spectral measurements by optical intensity changes at specific wavelength or optical spectral shifts. A custom smartphone multi-view App was developed to control the optical sensing parameters and to align each sample to the corresponding channel. The captured images were converted to the transmission spectra in the visible wavelength range from 400nm to 700nm with the high resolution of 0.2521nm per pixel. We validated the performance of this MSS via measuring the concentrations of protein and immunoassaying a type of human cancer biomarker. Compared to the standard laboratory instrument, the results sufficiently showed that this MSS can achieve the comparative analysis detection limits, accuracy and sensitivity. We envision that this multichannel smartphone optical biosensor will be useful in high-throughput point-of-care diagnostics with its minimizing size, light weight, low cost and data transmission function.

A Light-Emitting Diode- (LED-) Based Absorption Sensor for Simultaneous Detection of Carbon Monoxide and Carbon Dioxide.

A sensor was developed for simultaneous measurements of carbon monoxide (CO) and carbon dioxide (CO2) fluctuations in internal combustion engine exhaust gases. This sensor utilizes low-cost and compact light-emitting diodes (LEDs) that emit in the 3-5 µm wavelength range. An affordable, fast response sensor that can measure these gases has a broad application that can lead to more efficient, fuel-flexible engines and regulation of harmful emissions. Light emission from LEDs is spectrally broader and more spatially divergent when compared to that of lasers, which presented many design challenges. Optical design studies addressed some of the non-ideal characteristics of the LED emissions. Measurements of CO and CO2 were conducted using their fundamental absorption bands centered at 4.7 µm and 4.3 µm, respectively, while a 3.6 µm reference LED was used to account for scattering losses (due to soot, window deposits, etc.) common to the three measurement LEDs. Instrument validation and calibration was performed using a laboratory flow cell and bottled-gas mixtures. The sensor was able to detect CO2 and CO concentration changes as small as 30 ppm and 400 ppm, respectively. Because of the many control and monitor species with infra-red absorption features, which can be measured using the strategy described, this work demonstrates proof of concept for a wider range of fast (250 Hz) and low-cost sensors for gas measurement and process monitoring.

Zero order and signal processing spectrophotometric techniques applied for resolving interference of metronidazole with ciprofloxacin in their pharmaceutical dosage form.

Four rapid, simple, accurate and precise spectrophotometric methods were used for the determination of ciprofloxacin in the presence of metronidazole as interference. The methods under study are area under the curve, simultaneous equation in addition to smart signal processing techniques of manipulating ratio spectra namely Savitsky-Golay filters and continuous wavelet transform. All the methods were validated according to the ICH guidelines where accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can therefore be used for the routine analysis of ciprofloxacin in quality-control laboratories.

Stability-indicating methods for the analysis of ciprofloxacin in the presence of its acid induced degradation product: A comparative study.

Four rapid, simple, accurate and precise spectrophotometric methods were used for the determination of ciprofloxacin in the presence of its acidic degradation product. The methods under study are ratio derivative, ratio difference, mean centering and dual wavelength. All the methods were validated according to the ICH guidelines and the obtained accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can be used for the routine analysis of ciprofloxacin in quality-control laboratories.

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening.

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD+-coupled enzyme reaction. This dual-enzyme assay was used to determine Km and Vmax values of 104 microM and 5.7 nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

A simple method for determination of carmine in food samples based on cloud point extraction and spectrophotometric detection.

In this paper, a simple and cost effective method was developed for extraction and pre-concentration of carmine in food samples by using cloud point extraction (CPE) prior to its spectrophotometric determination. Carmine was extracted from aqueous solution using Triton X-100 as extracting solvent. The effects of main parameters such as solution pH, surfactant and salt concentrations, incubation time and temperature were investigated and optimized. Calibration graph was linear in the range of 0.04-5.0 µg mL(-1) of carmine in the initial solution with regression coefficient of 0.9995. The limit of detection (LOD) and limit of quantification were 0.012 and 0.04 µg mL(-1), respectively. Relative standard deviation (RSD) at low concentration level (0.05 µg mL(-1)) of carmine was 4.8% (n=7). Recovery values in different concentration levels were in the range of 93.7-105.8%. The obtained results demonstrate the proposed method can be applied satisfactory to determine the carmine in food samples.