Effect of redox label tether length and flexibility on sensor performance of displacement-based electrochemical DNA sensors

This article summarizes the sensor performance of four electrochemical DNA sensors that exploit the recently developed displacement-replacement sensing motif. In the absence of the target, the capture probe is partially hybridized to the signaling probe at the distal end, positioning the redox label, methylene blue (MB), away from the electrode. In the presence of the target, the MB-modified signaling probe is released; one type of probe is capable of assuming a stem-loop probe (SLP) conformation, whereas the other type adopts a linear probe (LP) conformation. Independent of the sensor architecture, all four sensors showed “signal-on” sensor behavior. Unlike the previous report, here we focused on elucidating the effect of the redox label tether length and flexibility on sensor sensitivity, specificity, selectivity, and reusability. For both SLP and LP sensors, the limit of detection was 10 pM for sensors fabricated using a signaling probe with three extra thymine (T3) bases linked to the MB label. A limit of detection of 100 pM was determined for sensors fabricated using a signaling probe with five extra thymine (T5) bases. The linear dynamic range was between 10 pM and 100 nM for the T3 sensors, and between 100 pM and 100 nM for the T5 sensors. When compared to the LP sensors, the SLP sensors showed higher signal enhancement in the presence of the full-complement target. More importantly, the SLP-T5 sensor was found to be highly specific; it is capable of discriminating between the full complement and single-base mismatch targets even when employed in undiluted blood serum. Overall, these results highlight the advantages of using oligo-T(s) as a tunable linker to control flexibility of the tethered redox label, so as to achieve the desired sensor response.     

基于聚苯乙烯微球放大的凝血酶化学发光检测新技术

本文以羧基96孔板为分离载体,核酸适配体作为分子特异性识别元件,聚苯乙烯微球作为放大载体,辣根过氧化物酶为标记物,构建了化学发光（CL）高灵敏度凝血酶检测新技术.实验结果表明：该放大技术不但灵敏度高,且抗干扰能力强,其他蛋白质如IgG、IgM、IgA、IgE、IFN均无明显干扰.聚苯乙烯微球放大体系中凝血酶的线性范围为7.8～250pmol/L,最低检测浓度可达3.9pmol/L;而不放大检测技术的线性范围为0.94～30nmol/L,最低检测浓度为0.46nmol/L,放大体系将检测灵敏度提高100多倍.综合而言,基于适配体识别和聚苯乙烯微球放大的凝血酶CL检测新技术具有通量大、简单快速和灵敏度高的特点,有望在凝血酶高通量检测领域获得应用.     

Pulsed multi-wavelength excitation using fiber-in-capillary light emitting diode induced fluorescence detection in capillary electrophoresis

A novel instrument was developed using a multi-wavelength pulsed LED array with in-column optic-fiber induced fluorescence detection by capillary electrophoresis. The light from 2 different wavelength LEDs (450 nm and 480 nm) was pulsed for short intervals at high intensity. The beam from each LED was collimated and reshaped with the gradient index (GRIN) lens group to achieve a highly effective coupling between LED light source and an optical fiber. The optical fiber was placed inside the capillary for in-capillary LED-induced fluorescence detection. The advantages of this system were validated by the simultaneous determination of vitamin B2 and fluorescein. Detection limits for vitamin B2 and fluorescein were estimated to be 5 nM and 0.29 nM (S/N = 3), respectively. The relative standard deviations (RSDs, n = 6) of the both compounds for migration time and peak area were better than 0.83%, 2.20% and 1.21%, 2.75%, respectively. The method was applied to the determination of vitamin B2 in commercial tablets and fluorescein in fluorescein sodium injection and the recoveries obtained were in the range of 96.6-102.0% and 99.9-102.8%, respectively. It was also applied to human serum, where the recoveries were found to be in the range of 94.4-97.0% and 92.6-96.4%, respectively. The system has been successfully applied in separation and determination of the both biological samples with acceptable analytical performance.     

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34 nM of HNE, and the response is linear in the tested concentration range of 0.34-68 nM with the detection limit of 47 pM.     

Direct determination of free bilirubin in serum at sub-nanomolar levels

Direct analysis of free bilirubin in human and animal blood serum samples is reported for the first time. A state-of-the-art system comprised of newly developed high-performance liquid chromatography (HPLC) on reverse-phase (RP) C18 support coupled with thermal lens spectrometric detection (TLS), based on excitation at λ = 457.9 nm by an argon laser was used for this purpose. This HPLC-TLS method enabled a baseline separation of all three structural isomers of bilirubin (XIII-α, IX-α and III-α) and the respective degradation products in isocratic mode in fewer than 7 min. The method excels in ultra-high sensitivity with limit of detection (LOD) and limit of quantitation (LOQ) of 90 pM and 250 pM, respectively. Moreover, this method also affords high precision and accuracy, with correlation coefficients R² > 0.997 over a broad linear range (0.250–150 nM) and R² = 0.9998 in a concentration range of clinical interest (0.500–25 nM). The method's boosted sensitivity enabled to streamline sample preparation to just one serum ultrafiltration step, which made qualitative evaluation of sample preparation possible for the first time. The performance of the HPLC-TLS method was assessed to have 20-fold enhanced sensitivity when compared to a comparable method incorporating HPLC coupled with diode array detector (DAD), which is also a novel method by itself, and could be applied for free bilirubin determination in patients with elevated bilirubin levels.     

Simultaneous detection for three kinds of veterinary drugs: Chloramphenicol, clenbuterol and 17-beta-estradiol by high-throughput suspension array technology

Suspension array technology for simultaneous detection of three kinds of veterinary drugs, chloramphenicol (CAP), clenbuterol and 17-beta-estradiol has been developed. Conjugates of chloramphenicol and clenbuterol coupled with bovine serum albumin were synthesized and purified. Probes of suspension array were constituted by coupling the three conjugates on the fluorescent microspheres/beads and the microstructures of the beads’ surface were observed by scanning electron microscopy which was a direct confirmation for the successful conjugates’ coupling. The optimal addition of conjugates and the amounts of antibodies were optimized and selected, respectively. Standard curves were plotted and the coefficient of determination-R² was greater than 0.989 which suggested good logistic correlation. The detection ranges for the three veterinary drugs are 40-6.25 × 10⁵ ng L⁻¹, 50-7.81 × 10⁵ ng L⁻¹ and 1 × 10^3-7.29 × 10⁵ ng L⁻¹, respectively and the lowest detection limits (LDLs) of them are 40, 50 and 1000 ng L⁻¹, respectively. The suspension array is specific and has no significant cross-reactivity with other chemicals. Meanwhile, unknown samples were detected by suspension array and ELISA in comparison with each other. The errors between found and real for the detection of the unknown samples were relatively small to both of the two methods, whereas, the detection ranges of suspension array are broader and sensitive than that of the traditional ELISA. The high-throughput suspension array is proved to be a novel method for multi-analysis of veterinary drugs with simple operation, high sensitivity and low cost.     

A gold nanoparticle labeling strategy for the sensitive kinetic assay of the carbamate-acetylcholinesterase interaction by surface plasmon resonance

The article presents a novel strategy for a sensitive investigation of the interaction between acetylcholinesterase (AChE) and its small molecular carbamate inhibitors. Two carbamate inhibitors with different ether linkages and the terminal lipoate were synthesized and labeled with gold nanoparticles (AuNPs). With the signal amplification of AuNPs, the specific interactions between the AuNPs labeled carbamate inhibitors (ALC1 and ALC2) and the immobilized AChE on sensor chip surface were readily examined. The detection sensitivities of ALC1 and ALC2 were 176 and 121 m°/nM, respectively, with the detection limits of 7.0 and 12 pM at a signal-to-noise ratio of 3. The association/dissociation constants for the binding interaction between carbamate inhibitors and AChE were reported for the first time. The affinity constants were estimated to be 3.13 × 10⁶ and 6.39 × 10⁵ M⁻¹ for ALC1 and ALC2 respectively. This AuNPs labeling strategy is versatile and may be applicable for the direct or competitive SPR kinetic assay of the interaction between small molecule inhibitors and their target proteins with a high sensitivity.     

Optimization of aptamer microarray technology for multiple protein targets

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl₂ and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.     

Investigation of plasma-functionalized multiwalled carbon nanotube film and its application of DNA sensor for Legionella pneumophila detection

A novel multiwall carbon nanotube (MWCNT) electrode functionalized with oxygen plasma treatment was prepared and characterized, and its DNA sensing ability for Legionella pneumophila (L. pneumophila) detection was examined using electrochemical measurement. A well-patterned MWCNT working electrode (WE) on a Pt track was fabricated using photolithography, transfer methods and an etching technique. The MWCNT WE was functionalized by oxygen plasma treatment prior to applying for DNA sensor. The surface morphology of the plasma-functionalized MWCNT (pf-MWCNT) WEs were observed by scanning electron microscope (SEM) and the change of chemical composition was characterized by X-ray photoelectron spectroscopy (XPS), and electrochemical measurements were performed using CV with ferricyanide/ferrocyanide redox couple. Effective areas of working electrodes were calculated to be 0.00453 cm² for pristine MWCNT electrode and 0.00747-0.00874 cm² for pf-MWCNT electrodes with different plasma treatment times. Differential pulse voltammetry (DPV) was carried out in methylene blue solution for DNA sensing. The pf-MWCNT based DNA sensor was successfully operated in a target concentration range of 10 pM to 100 nM and had a lower detection limit than a pristine MWCNT based DNA sensor.     

Ultrasensitive optical detection of trinitrotoluene by ethylenediamine-capped gold nanoparticles

This study found that 1,2-ethylenediamine (EDA) as a primary amine could be modified onto the surface of citrate-stabilized gold nanoparticles (Au NPs), and the EDA-capped Au NPs were successfully used as an ultrasensitive optical probe for TNT detection. The strong donor–acceptor (D–A) interactions between EDA and trinitrotoluene (TNT) at the Au NP/solution interface induced significant aggregation of the EDA-capped Au NPs, and enabled to easily realize the direct colorimetric detection of ultratrace TNT. The results showed that such a color change was readily seen by the naked eye, and the colorimetric detection could be down to 400 pM level of TNT with excellent discrimination against other nitro compounds. UV–vis absorption spectroscopy was used to examine the TNT-induced changes in local surface plasmon resonance (LSPR) of EDA-capped Au NPs, and a new LSPR band at ca. 630 nm arose along with the addition of TNT, which produced a detection limit of TNT down to ca. 40 pM. Furthermore, dynamic light scattering measurements evidenced the ultratrace TNT-induced small changes in the size of the EDA-capped Au NPs, and realized the quick and accurate detection of TNT in 0.4 pM level. These results demonstrated the ultrahigh sensitivity of this optical probe for TNT detection. Moreover, this optical probe is sample, stable, low-cost, and these excellent properties make it quite promising for infield and rapid detection of TNT.     

Adaptation of a load-inject valve for a flow injection chemiluminescence system enabling dual-reagent injection enhances understanding of environmental Fenton chemistry

Environmental Fenton chemistry has been poorly constrained within the marine environment at a multi-component level. A simple, unique, reconfiguration of a flow-injection analytical system combined with luminol chemiluminescence allows quasi-simultaneously the measurement, using a single load-inject valve and a single photon multiplier tube, of reduced iron, Fe(II), and hydrogen peroxide. The system enables rapid, every 22 s, measurements with good accuracy at environmentally relevant concentrations, less than 5% relative standard deviations on both a 5 nM Fe(II) standard and a 60 nM hydrogen peroxide standard. Limits of detection were as low as 40 pM Fe(II) and 100 pM hydrogen peroxide. The system showed excellent capability by measuring from within an organic rich seawater the photochemically induced production of Fe(II) and hydrogen peroxide and their subsequent cycling and Fenton like interactions.     

A highly selective turn-on fluorescent probe for hypochlorous acid based on hypochlorous acid-induced oxidative intramolecular cyclization of boron dipyrromethene-hydrazone

A BODIPY-based fluorescent probe, HBP, was developed for the detection of hypochlorous acid based on the specific hypochlorous acid-promoted oxidative intramolecular cyclization of heterocyclic hydrazone in response to the amount of HOCl. The reaction is accompanied by a 41-fold increase in the fluorescent quantum yield (from 0.004 to 0.164). The fluorescence intensity of the reaction between HOCl and HBP is linear in the HOCl concentration range of 1–8 μM with a detection limit of 2.4 nM (S/N = 3). Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HBP could be used as an effective fluorescent probe for detecting HOCl in living cells.     

A simple and rapid competitive enzyme-linked immunosorbent assay (cELISA) for high-throughput measurement of secretory immunoglobulin A (sIgA) in saliva

A simple competitive enzyme-linked immunosorbent assay (cELISA) was established for rapid measurement of secretory immunoglobulin A (sIgA) in saliva. The method was based on competitive reaction between the immobilized IgA and free IgA in the solution for the limited amount of horseradish peroxidase-conjugated rabbit anti-human IgA. In comparison with the conventionally used Sandwich ELISA, the cELISA is simpler, low-cost, and shows better reproducibility since it is not affected by the variation of capture antibodies from different batches. The assay time was also significantly reduced from more than 5 h to less than 3 h. Different curve-fitting models were compared, among which the fully specified logit-log model gave the best results. The linear working range and limit of detection were found to be 0.1-100 μg mL⁻¹ and 0.05 μg mL⁻¹, respectively. Matrix effects of saliva samples were investigated and a reasonable range of dilution factors were proposed. The developed method offers a very practical approach for high-throughput measurement of sIgA in saliva samples.     

Sensitivity of microarray based immunoassays using surface-attached hydrogels

A promising pathway to improve on the sensitivity of protein microarrays is to immobilize the capture antibodies in a three dimensional hydrogel matrix. We describe a simple method based on printing of an aqueous protein solution containing a photosensitive polymer and the capture antibody onto a plastic chip surface. During short UV-exposure photocrosslinking occurs, which leads to formation of a hydrogel, which is simultaneously bound to the substrate surface. In the same reaction the antibody becomes covalently attached to the forming hydrogel. As the capture antibodies are immobilized in the three-dimensional hydrogel microstructures, high fluorescence intensities can be obtained. The chip system is designed such, that non-specific protein adsorption is strongly prevented. Thus, the background fluorescence is strongly reduced and very high signal-to-background ratios are obtained (SBR > 6 for c_BSA = 1 pM; SBR > 100 for c_BSA > 100 pM). The kinetics of antigen binding to the arrayed antibodies can be used to determine the concentration of a specific protein (for example the tumor marker β₂-microglobulin) in solution for a broad range of analyte concentrations. By varying size and composition of the protein-filled hydrogel microstructures as well as adjusting the extent of labeling it is possible to easily adapt the surface concentration of the probe molecules such that the fluorescence signal intensity is tuned to the prevalence of the protein in the analyte. As a consequence, the signal tuning allows to analyze solutions, which contain both proteins with high (here: upper mg mL⁻¹ range) and with very low concentrations (here: lower μg mL⁻¹ range). This way quantitative analysis with an exceptionally large dynamic range can be performed.     

An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy

In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled “signal-on” and “signal-off” strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized “signal on” strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized “signal off” strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N = 3). In addition, this design strategy could be applied to the detection of other proteins and small molecules.     

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry

In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005 nM to 60 nM with a relatively low limit of detection of 1.7 pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.     

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)₃]²⁺ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1 pM of PDGF-BB. A calibration curve with a linearity range from 1 pM to 10 nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.     

A colorimetric method for protein assay via exonuclease III-assisted signal attenuation strategy and specific DNA–protein interaction

Taking advantage of exonuclease III (Exo III)-assisted signal attenuation strategy and the protection of DNA from Exo III-mediated digestion by specific DNA–protein interaction, a colorimetric method is proposed in this paper for protein assay. Specifically, in the absence of target protein, Exo III-assisted signal attenuation can be achieved by digesting the report DNA in a complex formed by the hybridization of a report DNA and a probe DNA. Nevertheless, in the presence of target protein, the binding of the analyte to the probe DNA will inhibit the Exo III-assisted nucleotides cleavage, so that cyclic signal attenuation is blocked. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, and the relationship can be shown by the surface plasmon changes caused by the report DNA-induced aggregation of DNA-modified gold nanoparticles (AuNPs). Our method can also have considerable sensitivity and selectivity, which has been demonstrated by the assay of human α-thrombin. Furthermore, by simply changing the sequence of the probe DNA, we can expand the application of our method to not only aptamer binding proteins but also DNA binding proteins, thus we have also used this method to analyze a specific serological marker for systemic lupus erythematosus (SLE) in this study. With a broad detection range of 1.3–133 nM and a detection limit of 0.61 nM (S/N = 3), it may hold great promise for clinical application.     

Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe3O4/polyaniline platform and multi-enzyme-functionalized carbon sphere label

An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe₃O₄/polyaniline/Nafion (Fe₃O₄/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab₂) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe₃O₄/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe₃O₄/polyaniline (Fe₃O₄/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8 pM and 2 nM with the detection limit of 4 pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants.     

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H₂O₂-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV–visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.