Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells.

Plastids are complex organelles that vary in size and function depending on the cell type. Accordingly, they can be referred to as amyloplasts, chloroplasts, chromoplasts, etioplasts, or proplasts, to only cite a few. Over the past decades, methods based on density gradients and differential centrifugation have been extensively used for the purification of plastids. However, these methods need large amounts of starting material, and hardly provide a tissue-specific resolution. Here, we applied our IPTACT (Isolation of Plastids TAgged in specific Cell Types) method, which involves the biotinylation of plastids in vivo using one-shot transgenic lines expressing the Translocon of the Outer Membrane 64 (TOC64) gene coupled with a biotin ligase receptor particle and the BirA biotin ligase, to isolate plastids from mesophyll and companion cells of Arabidopsis using tissue specific pCAB3 and pSUC2 promoters, respectively. Subsequently, a proteome profiling was performed, which allowed the identification of 1672 proteins, among which 1342 were predicted to be plastidial, and 705 were fully confirmed according to the SUBA5 database. Interestingly, although 92% of plastidial proteins were equally distributed between the two tissues, we observed an accumulation of proteins associated with jasmonic acid biosynthesis, plastoglobuli (e.g. NAD(P)H dehydrogenase C1, vitamin E deficient 1, plastoglobulin of 34 kDa, ABC1-like kinase 1) and cyclic electron flow in plastids originating from vascular tissue. Besides demonstrating the technical feasibility of isolating plastids in a tissue-specific manner, our work provides strong evidence that plastids from vascular tissue have a higher redox turnover to ensure optimal functioning, notably under high solute strength as encountered in vascular cells.

Tuning the Roundabout of Four-Point-Star Tiles with the Core Arm Length of Three Half-Turns for 2D DNA Arrays.

By rationally adjusting the weaving modes of point-star tiles, the curvature inherent in the tiles can be changed, and various DNA nanostructures can be assembled, such as planar wireframe meshes, perforated wireframe tubes, and curved wireframe polyhedra. Based on the weaving and tiling architectures for traditional point-star tiles with the core arm length at two DNA half-turns, we improved the weaving modes of our newly reported four-point-star tiles with the core arm length at three half-turns to adjust their curvature and rigidity for assembling 2D arrays of DNA grids and tubes. Following our previous terms and methods to analyze the structural details of E-tiling tubes, we used the chiral indices (n,m) to describe the most abundant tube of typical assemblies; especially, we applied both one-locus and/or dual-locus biotin/streptavidin (SA) labelling strategies to define the configurations of two specific tubes, along with the absolute conformations of their component tiles. Such structural details of the DNA tubes composed of tiles with addressable concave and convex faces and packing directions should help us understand their physio-chemical and biological properties, and therefore promote their applications in drug delivery, biocatalysis, biomedicine, etc.

Recent progress in analytical strategies of arsenic-binding proteomes in living systems.

Arsenic (As) is one of the most concerning elements due to its high exposure risks to organisms and ecosystems. The interaction between arsenicals and proteins plays a pivotal role in inducing their biological effects on living systems, e.g., arsenicosis. In this review article, the recent advances in analytical techniques and methods of As-binding proteomes were well summarized and discussed, including chromatographic separation and purification, biotin-streptavidin pull-down probes, in situ imaging using novel fluorescent probes, and protein identification. These analytical technologies could provide a growing body of knowledge regarding the composition, level, and distribution of As-binding proteomes in both cells and biological samples, even at the organellar level. The perspectives on analysis of As-binding proteomes are also proposed, e.g., isolation and identification of minor proteins, in vivo targeted protein degradation (TPD) technologies, and spatial As-binding proteomics. The application and development of sensitive, accurate, and high-throughput methodologies of As-binding proteomics would enable us to address the key molecular mechanisms underlying the adverse health effects of arsenicals.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Intracellular Protein Photoactivation Using Sterically Bulky Caging.

Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both in vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.

Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners.

Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each different randomized ssDNA library before embarking on any SELEX studies.

Immobilization of recombinant lysostaphin on nanoparticle through biotin-streptavidin conjugation technology as a geometrical progressed confrontation against Staphylococcus aureus infection.

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.

A flexible, robust microbead-based assay for quantification and normalization of target protein concentrations.

Although there are many methods for quantifying the concentration of specific proteins in samples, current techniques are technically challenging or do not easily lend themselves to normalization. Here, we describe a microbead-based assay for quantifying specific protein concentration(s) that is high-throughput, inexpensive, simple-to-use, and intrinsically incorporates normalization against the sample total protein content. This assay, termed the FRANC assay, exploits high affinity biotin-streptavidin binding to couple sample proteins to streptavidin-labelled magnetic microbeads. Proteins are then antibody-probed, followed by labeling of proteins on the microbead with fluorescent dye, and flow cytometry-based analysis. The FRANC assay demonstrates detection limits for target proteins in the femtogram range, with a linear range up to as much as 10 ng. Normalization of target protein concentrations resulted in an 80% reduction in variability as compared to non-normalized measurements. We conclude that the FRANC assay offers attractive advantages over current methods for quantifying specific protein(s) in samples.

Rolling circle amplification based colorimetric determination of Staphylococcus aureus.

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

Active Self-Assembly of Train-Shaped DNA Nanostructures via Catalytic Hairpin Assembly Reactions.

Biomolecular self-assembly is a powerful approach for fabricating supramolecular architectures. Over the past decade, a myriad of biomolecular assemblies, such as self-assembly proteins, lipids, and DNA nanostructures, have been used in a wide range of applications, from nano-optics to nanoelectronics and drug delivery. The method of controlling when and where the self-assembly starts is essential for assembly dynamics and functionalization. Here, train-shaped DNA nanostructures are actively self-assembled using DNA tiles as artificial "carriages," hairpin structures as "couplers," and initiators of catalytic hairpin assembly (CHA) reactions as "wrenches." The initiator wrench can selectively open the hairpin couplers to couple the DNA tile carriages with high product yield. As such, DNA nanotrains are actively prepared with two, three, four, or more carriages. Furthermore, by flexibly modifying the carriages with "biotin seats" (biotin-modified DNA tiles), streptavidin "passengers" are precisely arranged in corresponding seats. The applications of the CHA-triggered self-assembly mechanism are also extended for assembling the large DNA origami dimer. With the creation of 1D architectures established, it is thought that this CHA-triggered self-assembly mechanism may provide a new element of control for complex autonomous assemblies from a variety of starting materials with specific sites and times.

Development of a biotin-streptavidin-amplified nanobody-based ELISA for ochratoxin A in cereal.

A biotin-streptavidin-amplified enzyme-linked immunosorbent assay using a biotinylated nanobody (BA-Nb ELISA) was developed to detect ochratoxin A (OTA) in cereal. The limit of detection (LOD) of the BA-Nb ELISA, which equals to 10% maximal inhibitory concentration, was 0.011 ng/mL for OTA in buffer, and the sensitivity was approximately improved by one order of magnitude compared with the traditional Nb ELISA (LOD = 0.112 ng/mL). Under optimal conditions, the developed assay could be accomplished in 40 min with maximal inhibitory concentration of 0.138 ng/mL and the linear detection range of 0.034-0.460 ng/mL. The average recovery rate of the BA-Nb ELISA ranged from 92.8% to 114%, and the relative standard deviation was in the range of 2.04-9.85%. The developed BA-Nb ELISA was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the results indicated the reliability of BA-Nb ELISA for the detection of OTA in cereal.

Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection.

High-sensitivity C-reactive protein (hs-CRP) has been regarded as a risk predictor of cardiovascular disease (CVD). Despite there are many methods to detect hs-CRP, quantitative, rapid, convenient, multiplex and highly sensitive measurement of it is still a challenge for point-of-care applications. In this study, we developed a paper-based ELISA to detect hs-CRP and the sensitive chemiluminescence was applied as detection signal. In this developed assay method, CRP concentration and chemiluminescence intensity were linearly correlated (r = 0.999) with a limit of detection (LOD) as low as 0.49 ng mL-1, which was comparable to that of conventional ELISA and superior to most of the current reported POCT methods for detection of hs-CRP. The precision of the assay was confirmed for low coefficient of variations, less than 7% for intra-assay and less than 10% for inter-assay. In clinical sample analysis, the results of hs-CRP detected by this assay were in good accordance with which obtained by commercial high sensitivity ELISA kit for in vitro diagnosis (r = 0.975). This assay required only 4 µL of sample and could be finished in less than 30 min. It may therefore be employed as a rapid pre-screening tool to identify patients with elevated risk of CVD.

A voltammetric immunosensor for clenbuterol based on the use of a MoS2-AuPt nanocomposite.

An ultrasensitive immunosensor for the direct detection of the illegally used livestock feed clebuterol (CLB) is described. It is based on the use of a glassy carbon electrode modified with an MoS2-AuPt nanocomposite and on biotin-streptavidin interaction. The use of MoS2-AuPt accelerates electron transfer, and this leads to a sharp increase in the electrochemical signal for the electrochemical probe hydrogen peroxide. Differential pulse voltammetry was used to record the current signal at a peak potential of -0.18 V (vs SCE). Under optimal conditions, the electrode has a linear response in the 10 pg·mL-1 to 100 ng·mL-1 CLB concentration range and a 6.9 pg·mL-1 detection limit (based on the 3σ criterium). This immunosensor is sensitive, highly specific and acceptably reproducible, and thus represents a valuable tool for the determination of CLB in pork. Graphical abstract Schematic of a voltammetric immunosensor for the determination of clenbuterol (CLB) based on the use of a nanocomposite prepared from molybdenum disulfide and a gold-platinum alloy (MoS2-AuPt), and making use of the biotin-streptavidin system.

Electrochemical prostate specific antigen aptasensor based on hemin functionalized graphene-conjugated palladium nanocomposites.

An electrochemical aptasensor is described for the detection of prostate specific antigen (PSA). The aptasensor is based on the use of hemin-functionalized graphene-conjugated palladium nanoparticles (H-Gr/PdNPs) deposited on a glassy carbon electrode. The nanocomposites integrate the high electrical conductivity of graphene with the easily functionalized surface chemistry of PdNPs and their excellent catalytic property. The hemin placed on graphene acts as both a protective agent and an in-situ redox probe. The PdNPs provide numerous binding sites for the immobilization of DNA-biotin via coordinative binding between Pd and amino groups. A sensitive and specific PSA assay was attained by immobilizing the PSA aptamer via biotin-streptavidin interaction. The resulting aptasensor has a linear response that covers the PSA concentration range from 0.025 to 205 ng·mL-1, with a 8 pg·mL-1 lower detection limit (at -0.362 V, scan rate: 0.1 mV·s-1, S/N = 3). The method was applied to the quantitation of PSA in spiked serum samples, giving recoveries ranging from 95.0 to 100.3%. Graphical abstract A signal amplified and approving electrochemical aptasensor was constructed for the determination of prostate specific antigen (PSA) based on the use of hemin-functionalized graphene conjugated to palladium nanoparticles (H-Gr/PdNPs). The sensor has a wide linear range, a relatively low detection limit, satisfying stability and high specificity.

A fluorometric clenbuterol immunoassay based on the use of organic/inorganic hybrid nanoflowers modified with gold nanoclusters and artificial antigen.

Organic/inorganic hybrid nanoflowers were synthesized from calcium phosphate and protein modified fluorescent gold nanoclusters and antigens. These nanoflowers are shown to be well suited labels for bioassay because they fulfill the functions of biological recognition and signal output. A fluorometric immunoassay was developed that was combined with immunomagnetic separation. In the detection system, the red fluorescence of the supernatant (measured at excitation/emission wavelengths of 360/640 nm) is found to be proportional to the clenbuterol (Clen) concentration after two immunomagnetic separations. The assay has a linear response in the 0.5 µg L-1 to 40 µg L-1 Clen concentration range, and 0.167 µg L-1 limit of detection. This makes it well suited for food safety monitoring. The average recoveries from spiked samples range from 92.7 to 109.1% (intra-assay) and 101.2 to 125.7% (inter-assay) with relative standard deviations of <11.6%. Spiked swine urine samples were analyzed by this method, and the results correlated well with data obtained by LC-MS/MS. Graphical abstract Fluorescent hybrid nanoflowers were fabricated with gold nanoclusters (BSA-AuNCs) and antigens. A fluorometric immunoassay based on the use of such nanoflowers and based on immunomagnetic separation was developed to detect clenbuterol residues in swine urine with satisfactory recoveries and acceptable accuracy.

Affinity capture of aflatoxin B1 and B2 by aptamer-functionalized magnetic agarose microspheres prior to their determination by HPLC.

A novel adsorbent is described for magnetic solid-phase extraction (MSPE) of the aflatoxins AFB1 and AFB2 (AFBs). Magnetic agarose microspheres (MAMs) were functionalized with an aptamer to bind the AFBs which then were quantified by HPLC and on-line post-column photochemical derivatization with fluorescence detection. Streptavidin-conjugated MAMs were synthesized first by a highly reproducible strategy. They possess strong magnetism and high surface area. The MAMs were characterized by transmission electron microscopy, scanning electron microscopy, optical microscopy, laser diffraction particle size analyzer, Fourier transform infrared spectrometry, vibrating sample magnetometry and laser scanning confocal microscopy. Then, the AFB-aptamers were immobilized on MAMs through biotin-streptavidin interaction. Finally, the MSPE is performed by suspending the aptamer-modified MAMs in the sample. They are then collected by an external magnetic field and the AFBs are eluted with methanol/buffer (20:80). Several parameters affecting the coupling, capturing and eluting efficiency were optimized. Under the optimized conditions, the method is fast, has good linearity, high selectivity, and sensitivity. The LODs are 25 pg·mL-1 for AFB1 and 10 pg·mL-1 for AFB2. The binding capacity is 350 ± 8 ng·g-1 for AFB1 and 384 ± 8 ng·g-1 for AFB2, and the precision of the assay is <8%. The method was successfully applied to the analysis of AFBs in spiked maize samples. Graphical abstract Schematic of novel aptamer functionalized magnetic agarose microspheres (Apt-MAM) as magnetic adsorbents for simultaneous and specific affinity capture of aflatoxins B1 and B2 (AFBs).

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Indirect purification method provides high yield and quality ssDNA sublibrary for potential aptamer selection.

The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin-strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020±210nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary.

A heterogeneous biotin-streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples.

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. The small molecular TBC-hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA-ELISA were optimized. Under the optimal conditions, the limit of detection (IC10) was 0.0067 ng/ml and the median inhibitory concentration (IC50) was 0.66 ng/ml. Cross-reactivity values of the BA-ELISA with the tested TBC analogues were ⩽5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1-109.2%) were obtained. The results indicated that this proposed BA-ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring.