Rapid Colorimetric Assay Research Articles

Identification of antrodin B from Antrodia camphorata as a new anti-hepatofibrotic compound using a rapid cell screening method and biological evaluation.

Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) resulting from chronic liver diseases. Efficient and well-tolerated drugs for its treatment are urgently needed. This study aims to identify the active ingredients of Antrodia camphorata by a bioassay-guided fractionation approach and explore the acting mechanism by using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growth factor-β1 (TGF-β1). The accumulation of collagens was evaluated using chromogenic precipitation reaction with picro-sirius red (PSR) dye solution and quantified by spectrophotometric analysis of the dissolved stain. MTT assay, cell migration assay, quantitative polymerase chain reaction and western blotting analysis were used to determine the cell viability, cell migration and gene expression. We established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents. Stimulation with 10 ng/mL TGF-β1 for 48 h and 200 μL PSR dye solution were optimal for the colorimetric assay in CFSC-8B cells. We used SB431542, silybin and another 11 antifibrotic reagents to verify the cellular model. Within the safe doses, they attenuated ECM production induced by TGF-β1. Bioactivity-guided fractionation led to the identification of antrodin B from A. camphorata. Antrodin B significantly ameliorated cell proliferation, cell migration, suppressed HSC activation marker α-smooth muscle actin expression and ECM components Col1, Col3 and Fn expression, and blocked the phosphorylation of Smad2/3 induced by TGF-β1 in CFSC-8B cells in a dose-dependent manner. We developed a simple assay based on TGF-β1-induced total collagen accumulation in CFSC-8B cells and identified antrodin B which may serve as a potential candidate for treatment of liver fibrosis.

Rapid Colorimetric Detection of the Fungal Phytopathogen Synchytrium endobioticum Using Cyanine dye-Indicated PNA Hybridization

Synchytrium endobioticum is a parasitic fungus that is considered to be the most important potato pathogen worldwide. Spread of the fungus can result in catastrophic impacts to agriculture and economy, as it is very infective and can survive in spore form for over 70 years. To date, the gold standard for S. endobioticum identification relies on visual classification by highly trained personnel. This paper presents the colorimetric detection of S. endobioticum from potato wart samples using a peptide nucleic acid (PNA) probe and a cyanine dye. A segment of the 18S ribosomal RNA gene was sequenced for several S. endobioticum pathotypes and used to design a species-specific PNA probe. The probe was used in a rapid colorimetric hybridization assay that detected RNA from infected wart tissue down to 10−17 mol within 15 min. This technique demonstrates potential for rapid identification of potato wart in non-laboratory settings with minimally trained staff.

Auxin induces cell proliferation in an experimental model of mammalian renal tubular epithelial cells

Indole-3-acetic acid is the main auxin produced by plants and plays a key role in the plant growth and development. This hormone is also present in humans where it is considered as a uremic toxin deriving from tryptophan metabolism. However, beyond this peculiar aspect, the involvement of auxin in human pathophysiology has not been further investigated. Since it is a growth hormone, we evaluated its proliferative properties in an in vitro model of mammalian renal tubular epithelial cells. We employed an experimental model of renal tubular epithelial cells belonging to the LLC-PK1 cell line that is derived from the kidney of healthy male pig. Growth effects of auxin against LLC-PK1 cell lines were determined by a rapid colorimetric assay. Increasing concentrations of auxin (to give a final concentration from 1 to 1000 ng/mL) were added and microplates were incubated for 72 h. Each auxin concentration was assayed in four wells and repeated four times. Cell proliferation significantly increased, compared to control cells, 72 h after addition of auxin to cultured LLC-PK1 cells. Statistically significant values were observed when 100 ng/mL (p < 0.01) and 1000 ng/mL (p < 0.05) were used. In conclusion, auxin influences cell growth not only in plants, where its role is well documented, but also in mammalian cell lines. This observation opens new scenarios in the field of tissue regeneration and may stimulate a novel line of research aiming at investigating whether this hormone really influences human physiology and pathophysiology and in particular, kidney regeneration.

A Visual Colorimetric Probe for Naked-Eye Detection of Pamidronate Disodium in Human Plasma Based on Aggregation of Citrate-Capped Gold Nanoparticles

Herein, a sensitive, simple, and rapid colorimetric assay for the detection of pamidronate disodium was developed based on the aggregation of citrate-capped gold nanoparticles (AuNPs). This was exploited from the affinity of electron-rich atoms toward the surface of AuNPs, resulting in the aggregation of AuNPs through intermolecular hydrogen bonding interactions. As a result of aggregation under optimum conditions ([NaCl] = 25 mM, [AuNPs] = 3.3 nM and pH 7.0), the color of AuNPs was changed from red to blue and the plasmon band of AuNPs around 520 was decreased along with the formation of a new peak at a longer wavelength in the approximate range of 600 to 700 nm. The intensity ratios of absorbance at 650 nm to the original wavelength of 520 nm display a linear relation to the pamidronate disodium concentrations in the range of 50–225 μM. The detection limit (3σ) of this method was 43 μM. The proposed approach was successfully used for the determination of analyte in human plasma sample.

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts

The classical method to determine hydrogen peroxide (H2O2) scavenging activity of plant extracts is evaluated by measuring the disappearance of H2O2 at a wavelength of 230nm. Since this method suffers from the interference of phenolics having strong absorption in the UV region, a simple and rapid colorimetric assay was developed where plant extracts are introduced to H2O2, phenol and 4-aminoantipyrine reaction system in the presence of horseradish peroxidase (HRP). This reaction yields a quinoneimine chromogen which can be measured at 504nm. Decrease in the colour intensity reflects the H2O2 scavenged by the plant material.•Optimum conditions determined for this assay were 30min reaction time, 37°C, pH 7, enzyme concentration of 1U/ml and H2O2 concentration of 0.7mM. The limit of detection (LOD) and limit of quantitation (LOQ) were 136μM and 411μM, respectively.•Half maximal effective concentration required to scavenge 50% of H2O2 in the system (EC50 value) calculated for several plant extracts and standard antioxidants resulted in coefficient of variance (CV%) of the EC50 values less than 3.0% and correlation coefficient values (R2)>0.95 for all dose response curves obtained.•This method is convenient and very precise which is suitable for the rapid quantification of H2O2 scavenging ability of standard antioxidants and natural antioxidants present in plant extracts.

Colorimetric determination of total phenolic contents in ionic liquid extracts by paper microzones and digital camera

A rapid colorimetric paper microzone assay of total polyphenols in ionic liquid extracts was developed and validated.

Cytokine Impregnated Biomatrix: A New Tool to Study Multi-Wall Carbon Nanotubes Effects on Invertebrate Immune Cells

The novel features of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however they dissolved in water and accumulate in soils through the application of sewage sludge, accidental spills, and deposition from the air, agrochemicals or soil remediation. Given that several studies have revealed that chronic exposure to these nanomaterials products through the ingestion of drinking water, inhalation and dermal contact may harbour potential risks to human health, risk assessment of this nanomaterials in the aquatic environment are becoming essential. Here we propose a freshwater invertebrate, the leech Hirudo medicinalis, as a model to assess the effects MWCNTs on the immune system by means of in vivo and in vitro experiments. For this study, we used a consolidated experimental approach based on injection in the body wall of the leech of the biomatrice Matrigel (MG), added with a specific macrophage chemoattractant, the cytokine Allograft inflammatory factor-1 (AIF-1) and/or with MWCNTs. MG sponges analysis show the presence of a larger number of cells positive for both CD68 and HmAIF-1, specific monocyte-macrophage markers. Ultrastructural analysis suggests that MWCNTs may be internalized by phagocytosis but they seem also to be able to pierce cell membranes during cells migration. Cells extracted from MG were also used for in vitro treatment with MWCNTs at different concentration (2.5, 5, 10, 25, 50 and 100 μg/ml) for 24 h to study cell morphology changes and production of amyloid fibrils in order to encapsulate the foreign bodies. Our results, not only confirm the ability of MWCNTs in inducing a potent inflammatory response, but highlight rapid colorimetric assays that can be successfully used as sensitive tools for aquatic pollution biomonitoring.

Drug susceptibility testing of Mycobacterium tuberculosis by a nitrate reductase assay applied directly on microscopy-positive sputum samples

Aims and objectivesCurrent methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) are either costly or slow. As the prevalence of multidrug-resistant (MDR) strains increases, the need for fast, reliable, and inexpensive methods is obvious. This study evaluated a rapid colorimetric nitrate reductase assay (NRA) for direct DST of MTB directly from clinical sputum samples. MethodsA total of 111 sputa with positive microscopy results for acid-fast bacilli (AFB) with more than 10 AFB per high-power field were used in the study. The samples were decontaminated using the modified Petroff method. The NRA results were compared with the reference indirect proportion method. ResultsThe sensitivity and the specificity of the direct NRA were 90% and 97.3%, 92.6% and 98.2%, 52.9% and 100%, and 28.6% and 100% for rifampin, isoniazid, streptomycin, and ethambutol, respectively. The results were in most cases available in 28days (84.3%). ConclusionsThe direct NRA could be used as a rapid, inexpensive, and accurate method to determine rifampin and isoniazid susceptibility directly from sputum. The technique might become a valid alternative to traditional methods, especially in low-income countries.

Microfluidic toner-based analytical devices: disposable, lightweight, and portable platforms for point-of-care diagnostics with colorimetric detection.

This chapter describes the development of microfluidic toner-based analytical devices (μTADs) to perform clinical diagnostics using a scanner or cell-phone camera. μTADs have been produced in a platform composed of polyester and toner by the direct-printing technology (DPT) in a matter of minutes. This technology offers simplicity and versatility, and it does not require any sophisticated instrumentation. Toner-based devices integrate the current generation of disposable analytical devices along paper-based chips. The cost of one μTAD has been estimated to be lower than $0.10. In addition, these platforms are lightweight and portable thus enabling their use for point-of-care applications. In the last 5 years, great efforts have been dedicated to spread out the use of μTADs in bioassays. The current chapter reports the fabrication of printed microplates and integrated microfluidic toner-based devices for dengue diagnostics and rapid colorimetric assays with clinically relevant analytes including cholesterol, triglycerides, total proteins, and glucose. The use of μTADs associated with cell-phone camera may contribute to the health care, in special, to people housed in developing regions or with limited access to clinics and hospitals.

Intrinsic enzyme mimicking activity of gold nanoclusters upon visible light triggering and its application for colorimetric trypsin detection

In this research, a novel enzyme mimetics based on the photochemical property of gold nanoclusters was demonstrated. It was found that the bovine serum albumin (BSA) stabilized red or blue emitting gold nanoclusters (Au NCs) exhibited enzyme-like activity under visible light irradiation. The BSA–Au NCs had better stability against stringent conditions compared to natural enzyme. In addition, the photostimulated enzyme mimetics of BSA–Au NCs showed several unprecedented advantages over natural peroxidase or other existing alternatives based on nanomaterials, such as the independence of hydrogen peroxide on activity and the easily regulated activity by light irradiation. The mechanism of the photoresponsive enzyme-like activity of BSA–Au NCs was investigated. The photoactivated BSA–Au NCs was designed to develop a facile, cheap, and rapid colorimetric assay to detect trypsin through trypsin digestion of the protein template of BSA-stabilized Au NCs. The limit of detection for trypsin was 0.6μg/mL, which was much lower than the average level of trypsin in patient′s urine or serum.

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

Evaluation of a rapid colorimetric assay for detection of bacterial contamination in apheresis and pooled random‐donor platelet units

Despite existing strategies, bacterial contamination of platelets (PLTs) remains a problem, and reliable testing near the time of use is needed. We evaluated the BacTx assay (Immunetics, Inc.), a rapid colorimetric assay for detection of bacterial peptidoglycan, for this purpose. Apheresis- and whole blood-derived PLT units, the latter tested in 6-unit pools, inoculated with 10 representative bacterial species (eight aerobic, two anaerobic), were tested with the BacTx assay at two sites to determine analytic sensitivity and time to detection. Specificity on sterile PLTs and reproducibility across different PLT units and assay kit lots was also determined. Analytical sensitivity for the 10 bacterial species ranged from 6.3 × 10(2) to 7.6 × 10(4) colony-forming units (CFUs)/mL. In time-to-detection studies after inoculation of PLTs with 0.7 to 5.3 CFUs/mL, 10 replicates of all eight aerobic species were positive when bacterial titers were above the analytic sensitivity detection limit, which occurred at 48 hours for 60 PLT units and at 72 hours for the remaining 4 units, as well as at 7 days for all units. Specificity was 99.8% and reproducibility was 100%. The BacTx assay had an analytical sensitivity below the 10(5) CFUs/mL threshold of clinical significance, detected all eight aerobic bacterial species 48 to 72 hours after inoculation as well as at 7 days, and had high specificity and reproducibility. These findings suggest that the BacTx assay will be a valuable test for detection of clinically relevant levels of bacterial contaminants in PLT units and pools near time of use.

The novel histone deacetylase inhibitor thailandepsin A inhibits anaplastic thyroid cancer growth

BackgroundAnaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. MethodsHuman-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. ResultsTDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. ConclusionsTDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

Thermally treated bare gold nanoparticles for colorimetric sensing of copper ions

We demonstrate a sensitive and rapid colorimetric assay for selective detection of copper ions based on the strong coordination between Cu(II) ions and the tetrahydroxyaurate anions [Au(OH)4]− on the surface of thermally treated bare gold nanoparticles (GNPs). The method for making the unmodified GNPs is simple and results in a nanomaterial with a highly specific response to Cu(II). The thermal treatment of the bare GNPs and the recognition of Cu(II) ions is accomplished in a single step within 5 min. The presence of Cu(II) causes the color to change from red to purple-blue. The limit of detection (LOD) is 0.04 μM of Cu(II) when using UV–vis spectrometry and ratioing the absorbances at 650 and 515 nm, respectively. The method also is amenable to bare eye (visual) inspection and in this case has an LOD of 2.0 μM of Cu(II).

In vitro activity of ABT-888 (A), 5-FU (F), and dacarbazine (D) in neuroendocrine tumors (NET).

236 Background: Low-grade neuroendocrine tumors (NET) have few cytotoxic chemotherapy options. Data suggests that a combination of temozolomide and a fluoropyrimidine has clinical efficacy. ABT-888 is a novel poly-ADP ribose polymerase (PARP) inhibitor that has been tested in a phase 1 setting with temozolomide, with synergy demonstrated in preclinical models of other tumors. We proposed an in-vitro study of ABT-888 with varying concentrations of dacarbazine (D), and 5-FU (F) on human neuroendocrine cells (BON). Methods: BON cells were incubated with varying concentrations and combinations of ABT-888 (2.5, 5 and 10uM), F (25-100uM), and D (25-100uM) for 96 hours. After incubation, cell growth was measured by MTT rapid colorimetric assay with absorbance reported as mean % control. Western blot analysis was performed for chromogranin A, PARP, γH2AXand XIAP to assess for cell death and on-target effects for the population treated with 5-FU. Combination indices (CI) were calculated using the Chou-Talalay method using CompuSyn. CI’s below 1 signified synergy. Results: ABT-888 alone did not demonstrate any antitumor effect (103%). D alone had antitumor effect (74% at 50uM, 67% at 100uM) which was improved by adding ABT-888 (70% at 50uM D; CI 0.73; p=0.06, 60% at 100 uM D; CI 0.88; p=0.0003). F alone had antitumor effect (82% at 50uM and 71% at 100uM) which was improved by adding 2.5uM ABT-888 (71% at 50uM of F; CI=0.59, and 58% at 100uM of F; CI=0.74), and further enhanced with 5uM of concomitant ABT-888 (56% at 50uM of F; CI=0.68, and 49% at 100uM of F; CI=0.76). Western analysis of lysates showed markers of increased apoptosis, decreased PARP, and decreased expression of CgA. The combination of F+D did not demonstrate increased cytotoxicity with the addition of ABT-888 (58% with/without ABT-888 p=0.61). Conclusions: ABT-888 demonstrated in vitro synergy against BON cells with F or D. The combination of all three compounds (A+F+D) did not demonstrate synergy above F+D. Synergy was statistically significant with increasing doses of cytotoxic compounds which are achievable in vivo with current doses of A, F, and D. The combination of ABT-888 with temozolomide or a fluoropyrimidine merits further study in human clinical trials.

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography–mass spectrometry (GC–MS).In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of kcat/KM among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles

In this paper, we developed a simple and rapid colorimetric assay for protein detection based on the reduction of dye molecules catalyzed by silver nanoparticles (AgNPs). Aptamer-modified magnetic particles and aptamer-functionalized AgNPs were employed as capture and detection probes, respectively. Introduction of thrombin as target protein could form a sandwich-type complex involving catalytically active AgNPs, whose catalytic activity was monitored on the catalytic reduction of rhodamine B (RhB) by sodium borohydride (NaBH4). The amount of immobilized AgNPs on the complex increased along with the increase of the thrombin concentration, thus the detection of thrombin was achieved via recording the decrease in absorbance corresponding to RhB. This method has adopted several advantages from the key factors involved, i.e., the sandwich binding of affinity aptamers contributed to the increased specificity; magnetic particles could result in rapid capture and separation processes; the conjugation of AgNPs would lead to a clear visual detection. It allows for the detection limit of thrombin down to picomolar level by the naked eye, with remarkable selectivity over other proteins. Moreover, it is possible to apply this method to the other targets with two binding sites as well.

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A(260)), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).(1) Though it cannot readily distinguish RNA from DNA, the A(260)/A(280) ratio is commonly employed, as it offers a simple and rapid(2) assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.5(3). However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ≈280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A(260)/A(280) ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.53. However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ≈280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).