Sub-nanogram Levels Research Articles

Improved Data Acquisition Settings on Q Exactive HF-X and Fusion Lumos Tribrid Orbitrap-Based Mass Spectrometers for Proteomic Analysis of Limited Samples.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Targeting altered glycosylation in secreted tumor glycoproteins for broad cancer detection.

There is an urgent need to develop new tumor biomarkers for early cancer detection, but the variability of tumor-derived antigens has been a limitation. Here we demonstrate a novel anti-Tn antibody microarray platform to detect Tn+glycoproteins, a near universal antigen in carcinoma-derived glycoproteins, for broad detection of cancer. The platform uses a specific recombinant IgG1 to the Tn antigen (CD175) as a capture reagent and a recombinant IgM to the Tn antigen as a detecting reagent. These reagents were validated by immunohistochemistry in recognizing the Tn antigen using hundreds of human tumor specimens. Using this approach, we could detect Tn+glycoproteins at subnanogram levels using cell lines and culture media, serum, and stool samples from mice engineered to express the Tn antigen in intestinal epithelial cells. The development of a general cancer detection platform using recombinant antibodies for detection of altered tumor glycoproteins expressing a unique antigen could have a significant impact on cancer detection and monitoring.

Detection methods for sub-nanogram level of emerging pollutants - Per and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are organofluorine compounds has been manufactured for more than five decades and used in different purposes. Among persistent organic pollutants, PFAS are toxic, bioaccumulative in humans, wildlife, and global environment. As per environmental protection agency (EPA) guidelines, the perfluorooctanoate and perfluorooctane sulfonate permissible limit was 0.07ng/L in drinking water. When the concentration exceeds the acceptable limit, it has negative consequences for humans. In such a case, PFAS monitoring is critical, and a quick detection technique are highly needed. Health departments and regulatory agencies have interests in monitoring of PFAS presences and exposures. For the detection of PFAS, numerous highly precise and sensitive chromatographic methods are available. However, the drawbacks of analytical techniques include timely sample preparations and the lack of on-site applicability. As a result, there is an increasing demand for simple sensor systems for monitoring of PFAS in real field samples. In this review, we first describe the sample pre-treatment and analytical techniques for the detection of PFAS. Second, we broadly discussed available sensor system for the quantification of PFAS in different filed samples. Finally, future trends in PFASs sensor are also presented.

Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples.

In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein- and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.

Precise determination of lithium isotope ratios at the sub-nanogram level by QQQ-ICP-MS: application to natural waters and carbonates

Lithium isotopes are a powerful tracer of low and high temperature geochemical processes. Utilizing commonly available instrumentation (QQQ-ICP-MS), we have developed a novel method for precise determination of Li isotopes from sub-nanogram samples.

Novel simple enzymatic potentiometric approach for toxicological assessment of anticholinesterase and Alzheimer’s drugs Enzymatic approach toxicological assessment

Background and objectives A simple and sensitive enzymatic potentiometric biosensor has been developed for toxicological studies of anticholinesterase drugs based on their inhibitory effect on cholinesterase activity. To verify the applicability, eight pharmaceutical formulations for Alzheimer's treatment were assayed, namely rivastigmine, pyridostigmine, cyclopentolate, memantine, meclofenoxate, carbamazepine, oxfendazole, and methotrexate.Materials and methods Disposable screen-printed potentiometric sensors were utilized for monitoring the cholinesterase activity. The reaction conditions including the optimal enzyme substrate, incubation periods, and the linearity range for each drug were optimized for each drug.Results and conclusion Different sensitivities within subnanogram levels were reported based on the inhibitory effect of the aforementioned pharmaceutical compounds and their LD50 value. The proposed method showed improved sensitivity for the investigated compounds compared with their reported electroanalytical approaches. The introduced analysis protocol was successfully utilized for assaying the cited drugs in their pharmaceutical and environmental samples using a portable measuring system. Moreover, the toxicity of the pharmaceutical compounds against in-vitro cholinesterase enzymes studies can be performed with simple instrumentation requirements.

Development of a Simple and Powerful Analytical Method for Formaldehyde Detection and Quantitation in Blood Samples.

Human beings are easily exposed to formaldehyde (FA) in a living environment. Entry of FA into the human body can have adverse effects on human health, depending on the FA concentration. Thus, a quantitative analysis of FA in blood is necessary in order to estimate its effect on the human body. In this study, a simple and rapid analytical method for the quantitation of FA in blood was developed. The total analysis time, including the pretreatment procedure, was less than 20 min. To ensure a stable analysis, blood samples were stabilized using tripotassium ethylenediaminetetraacetic acid solution, and FA was selectively derivatized using 2,4-dinitrophenylhydrazine as pretreatment procedures. The pretreated samples were analyzed using a high-performance liquid chromatography-UV system, which is the most common choice for analyzing small-molecule aldehydes like formaldehyde. Verification of the pretreatment methods (stabilization and derivatization) using FA standards confirmed that the pretreatment methods are highly reliable in the calibration range 0.012–5.761 ng μL–1 (slope = 684,898, R2 = 0.9998, and limit of detection = 0.251 pg·μL–1). Analysis of FA in the blood samples of a Yucatan minipig using the new method revealed an average FA concentration of 1.98 ± 0.34 ng μL–1 (n = 3). Blood samples spiked with FA standards were analyzed, and the FA concentrations were found to be similar to the theoretical concentrations (2.16 ± 0.81% difference). The method reported herein can quantitatively analyze FA in blood at a sub-nanogram level within a short period of time and is validated for application in blood analysis.

Nanochannel-driven rapid capture of sub-nanogram level biomarkers for painless preeclampsia diagnosis

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome recognized as the leading cause of maternal and fetal morbidity and mortality worldwide. Painful blood-collection procedures or low accuracy of non-invasive approaches require faster, patient-friendly, and more sensitive diagnostic technologies. Here we report a painless, highly sensitive detection platform using nanoporous microneedles (nMNs) that enables rapid capture of biomarkers present at sub-nanogram levels. The highly porous nanostructures on the nMN surface were prepared by anodization of aluminum MN and then functionalized by immobilization of capture antibodies to detect target biomarkers based on an immunoassay method. The immuno-functionalized nMN array demonstrated rapid capture of an estrogen (E2) biomarker for PE following a 1-min incubation and exhibited a concentration-dependent change in fluorescence intensity over the E2 range of 0.5 ng mL−1 to 1000 ng mL−1 after treatment with fluorescence-detection antibodies. Remarkably, the nMN patch selectively detected sub-nanogram-levels of E2 in subcutaneous interstitial fluid from rats with increased diagnostic accuracy as compared with commercial immunoassay kits. This bio-functionalized nMN platform showed improved biosensing capability for multiple PE-related biomarkers, including hormones and proteins. Furthermore, this painless method demonstrated efficacy as a point-of-need diagnostic platform using portable smartphone-based fluorescence microscope to obtain fluorescence images of biomarker-captured nMN arrays.

Accurate and precise determination of Lu and Hf contents and Hf isotopic composition at the sub-nanogram level in geological samples using MC-ICP-MS

We describe protocols for highly reproducible analysis of Lu and Hf contents and Hf isotopic composition at sub-nanogram levels in geological samples by MC-ICP-MS.

Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis.

BackgroundNearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.MethodsSerum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed.ResultsIntense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin [ACG]) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin [ABA], and Amaranthus caudatus agglutinin [ACA]) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation.ConclusionsThis is the first report of a glycoprotein biomarker using glycan change at a local lesion to assess disease activity in autoimmune diseases. Differences in the degree of serum MMP-3 α-2,6-sialylation may be a useful index for estimating disease activity.

High-Sensitivity and High-Efficiency Detection of DNA Hydroxymethylation in Genomic DNA by Multiplexing Electrochemical Biosensing

DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification. The 5-hmC was first glycosylated by T4 β-glucosyltransferase and then oxidated by sodium periodate. The resulting glucosyl-modified 5-hmC (5-ghmC) was incubated with ARP-biotin and was bound to avidin-HRP. The 5-hmC can be detected at the subnanogram level. Finally, we performed 5-hmC detection for mouse tissue samples and cancer cell lines. The limit of detection of the MEC biosensor is 20 times lower than that of commercial kits based on optical meaurement. Also, the biosensor presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at any other unhydroxymethylated nucleic acid bases. Importantly, benefited by its multiplexing capacity, the developed MEC biosensor showed excellent high efficiency, which was time-saving and cost less.

Silver nanoislands on cellulose fibers for chromatographic separation and ultrasensitive detection of small molecules.

High-throughput small-molecule assays play essential roles in biomedical diagnosis, drug discovery, environmental analysis, and physiological function research. Nanoplasmonics holds a great potential for the label-free detection of small molecules at extremely low concentrations. Here, we report the development of nanoplasmonic paper (NP-paper) for the rapid separation and ultrasensitive detection of mixed small molecules. NP-paper employs nanogap-rich silver nanoislands on cellulose fibers, which were simply fabricated at the wafer level by using low-temperature solid-state dewetting of a thin silver film. The nanoplasmonic detection allows for the scalable quantification and identification of small molecules over broad concentration ranges. Moreover, the combination of chromatographic separation and nanoplasmonic detection allows both the highly sensitive fluorescence detection of mixed small molecules at the attogram level and the label-free detection at the sub-nanogram level based on surface-enhanced Raman scattering. This novel material provides a new diagnostic platform for the high-throughput, low-cost, and label-free screening of mixed small molecules as an alternative to conventional paper chromatography.

Rapid detection and isotopic measurement of discrete inorganic samples using acoustically actuated droplet ejection and extractive electrospray ionization mass spectrometry

The rapid detection, screening, and isotopic signature analysis of inorganics provide invaluable information for a variety of applications including explosive device detection, nuclear forensics, and environmental monitoring. The coupling of ultrasonic nebulization and extractive electrospray ionization (EESI) enabled the mass spectrometric (MS) detection and analysis of inorganics from microliter sample solution aliquots. Ultrasonic nebulization and acoustic pressure wave focusing within an array of exponential horn structures were utilized for the efficient atomization of discrete liquid samples ranging in volume from 3 μL to 10 μL pipetted aliquots. In conjunction with an electro-flow focusing source for extractive electrospray ionization (EESI), in-source collision-induced dissociation (CID) was utilized to enhance inorganic detection through fragmentation of adducts and reduction in chemical noise from organic compounds. The investigated system enhanced detection of the singly charged elemental cation species and provided accurate measurements of isotopic distributions for a number of metal ions. The extent of CID demonstrated the competition between ligand loss from hydrate clusters and charge reduction from the doubly charged to singly charged cations for the alkaline earth metal ions of strontium and barium. Inorganics were also detected from complex matrices, including synthetic fingerprint material and sediment, without detriment to device operation. The described system provides a versatile tool for the rapid detection, speciation, and isotopic identification of inorganic compounds at nanogram to sub-nanogram levels from microliter aliquots. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Chemically functionalized carbon films for single molecule imaging

Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.

Sensitive detection of black powder by stand-alone ion mobility spectrometer with chlorinated hydrocarbon modifiers in drift gas

This paper introduces a simple method for selective and sensitive detection of black powder by adding chlorinated hydrocarbons in the drift gas instead of changing the structure of conventional ion mobility spectrometer (IMS). The function of chloride modifiers was to substitute Cl(-)(H₂O)n for [O₂⁻ (H₂O)(n)] in the drift region so as to avoid the overlap between O₂⁻ (H₂O)(n) and sulfur ion peaks. Among CH₂Cl₂, CHCl₃ and CCl₄, CCl₄ was chosen as the modifier due to the best peak-to-peak resolution and stability towards the fluctuation of modifier concentration. With 1.4 ppm CCl₄ as the modifier, the minimum detectable quantity of 0.1 ng for sulfur was achieved. Moreover, this method showed the ability for detection of common explosives at sub-nanogram level, such as black powder (BP), ammonium nitrate fuel oil (ANFO), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN). In summary, this method requiring no configuration modification has high sensitivity and selectivity, and consumes trace amount of modifier. And these characteristics make it easy to be adopted in current deployed IMS to detect black powder explosives.

Multi-Element Analysis by Portable Total Reflection X-ray Fluorescence Spectrometer

Multi-element solutions containing the 11 elements S, K, Sc, V, Mn, Co, Cu, Ga, As, Br and Y were analyzed by a portable total reflection X-ray fluorescence (TXRF) spectrometer. The excitation parameters (glancing angle, operational voltage and current) and sample amount were optimized for the portable TXRF in order to realize the smallest possible detection limits for all elements. The excitation parameter dependencies of the fluorescence signal and background for the detected elements are explained in detail. Background contributed by the sample carrier is also discussed. Consequently, nine elements were detectable at sub-nanogram levels in a single measurement of 10 min under the optimal experimental conditions. The portable TXRF spectrometer was found to be suitable for simultaneous multi-element analysis with low detection limits. The features of high sensitivity, small sample amount required, and fast detection of a wide range of elements make the portable TXRF a valuable tool in various applications, such as field studies in environmental and geological investigations.

Application of nanobalance technique and principal component analysis for detection of the soil fumigant Telone residues in the air

Quartz crystal nanobalance (QCN) technique is considered as a powerful mass sensitive sensor for monitoring of materials in the sub-nanogram level. In the current study, a method based on QCN technique developed to determine Telone in air. Various coating materials including methyl phenyl silicon, 75% phenyl (OV25) and molecularly imprinted polymer (MIP) were employed. The frequency shift of OV25-modified quartz crystal was found to be linear against organohalogen compounds [Telone (soil fumigant), Koril (Herbicide), Endosulfan (organochlorine insecticide) and Chloroform (solvent)] concentrations in the range of 2.4 to 48 mg L−1 for Telone vapor and 4.8–24 mg L−1 for three other vapors. The correlation coefficients for Telone, Koril, Endosulfan and Chloroform were 0.992, 0.996, 0.989 and 0.991, respectively. The principal component analysis was also utilized to process the frequency response data of the organic vapors. Using principal component analysis, it was found that more than 93.85% of the data variance could still be explained by use of two principal components (PC1 and PC2). Subsequently, the successful discrimination of Telone and other compounds was quite possible through the principal component analysis of the transient responses of the OV25-modified electrode. In the second method, a molecularly imprinted polymer-coated sensor for Telone was developed. Molecularly imprinted polymer coated quartz crystal (MIP-QCN) showed a selective response to Telone and gave a linear relationship between frequency shift and amount of Telone from 1 to 48 mg L−1. In this investigation, the proficiency of MIP-QCN and OV25-modified QCN sensors were compared.

SIMULTANEOUS ESTIMATION OF RAMIPRIL AND ITS ACTIVE METABOLITE RAMIPRILAT IN HUMAN PLASMA BY ESI-LC-MS/MS

A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the simultaneous determination of ramipril and ramiprilat in human plasma. The solid-phase extraction technique was used for the extraction of ramipril and ramiprilat from human plasma. Enalapril was used as the internal standard (IS).Chromatography was performed on a Aquasil C18, 100mm×2.1mm, 5µ column, with the mobile phase consisting of acetonitrile-deionsed water (in a 65:35 ratio) and 1.0mLL-1 ammonium trifluoroacetate solution (1.0M), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-nanogram levels. The method was validated and the lower limit of quantification for ramipril and ramiprilat was found to be 0.1 ngmL−1and 0.1 ngmL−1, respectively. The mean recovery for ramipril and ramiprilat ranged from 63.5 to 74.3%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of ramipril and ramiprilat using single IS in a single experiment for bioequivalence studies, with a chromatographic run time of 3.0 min only.

Application of green synthesis of gold nanoparticles for sensitive detection of aflatoxin B1 based on metal enhanced fluorescence

A green synthesis approach is reported for the synthesis of gold nanoparticles (GNPs) using the leaf extract of Coreopsis lanceolata. The synthesized GNPs were effectively utilized in the detection of aflatoxins at low concentrations based on the metal enhanced fluorescence principle. The structural characteristics of the GNPs were analyzed by UV-Vis spectroscopy, TEM and spectrofluorimetry. Bioreduction of auric chloride by C. lanceolata leaf extract resulted in the synthesis of spherical, homogenous GNPs. Further GNPs were conjugated with anti-aflatoxin antibodies and aflatoxin B1 to study the metal enhanced charge transfer between the nanoparticles and aflatoxins, which can be effectively utilized in the detection of aflatoxins at very low concentrations. The studies showed up to a 10-fold increase in the fluorescence intensity of aflatoxin B1 at picogram levels. Linearity was found to be in the range of 10 picogram to 500 picogram with a regression coefficient R2 = 0.970. The limit of detection was found to be 5 picogram. Thus the fluorescent GNPs synthesized by this green route can be effectively used in the detection of aflatoxins at sub-nanogram levels by utilizing the metal enhanced fluorescence principle.

Functionalized Ultrathin Carbon Films for Imaging Low-Abundance Biological Complexes

By single particle electron cryo-microscopy (cryoEM), structures of low-abundance biological complexes can be obtained if the surface density of the complexes on the EM grids can be made sufficiently high for data collection. In order to prevent most of the molecules from being lost during specimen preparation, new methods are needed to retain them selectively to the surface. Such methods will permit cryoEM imaging of macromolecules that are available at subnanogram levels. Available methods along this line of thinking all add significant biomass to the background and are not so simple or robust for routine operations. We have developed a novel “bottom-up” approach to selectively anchor proteins of interest onto the surface of nanometer-thick carbon films that are routinely used for cryoEM imaging. We show that by chemically modifying the carbon films, specific ligands can be introduced onto carbon surfaces in order to enrich complexes bearing appropriate functional groups. In particular, we have been developing the chemical procedures that present on the carbon films specific ligands for His-tagged, GST-tagged and oligonucleotide-binding complexes as well as for immunoglobulin molecules. Meanwhile, we are applying this technology to the structural study of important low-abundance biological complexes by single particle EM. Our results demonstrate that the surface-engineered carbon films should have general applicability to a wide range of biophysical and biomedical problems.