Diluted Serum Samples Research Articles

Cellular and Humoral Immune Responses and Breakthrough Infections After Two Doses of BNT162b Vaccine in Healthcare Workers (HW) 180 Days After the Second Vaccine Dose.

BackgroundImmunity and clinical protection induced by mRNA vaccines against SARS-CoV-2 have been shown to decline overtime. To gather information on the immunity profile deemed sufficient in protecting against hospitalization, we tested IgG levels, interferon-gamma (IFN-γ) secretion, and neutralizing antibodies 180 days (d180) after the second shot of BNT162b vaccine, in HW.MethodsA total of 392 subjects were enrolled. All received BioNTech/Pfizer from February 2020 to April 2021. The vaccine-specific humoral response was quantitatively determined by testing for IgG anti-S1 domain of SARS-CoV-spike protein. Live virus microneutralization (MN) was evaluated by an assay performing incubation of serial 2-fold dilution of human serum samples, starting from 1:10 to 1:5120, with an equal volume of Wuhan strain and Delta VOC viral solution and assessing the presence/absence of a cytopathic effect. SARS-CoV-2-spike protein-specific T-cell response was determined by a commercial IFN-γ release assay.ResultsIn 352 individuals, at d180, IgG levels decreased substantially but no results below the assay's positivity threshold were observed. Overall, 22 naive (8.1%) had values above the highest threshold. Among COVID-naive, the impact of age, which was observed at earlier stages, disappeared at d180, while it remained significant for 81 who had experienced a previous infection. Following the predictive model of protection by Khoury, we transformed the neutralizing titers in IU/ml and used a 54 IU/ml threshold to identify subjects with 50% protective immunity. Overall, live virus MN showed almost all subjects with previous exposure to SARS-CoV-2 neutralized the virus as compared to 33% of naive double-dosed subjects (p < 0.0001). All previously exposed subjects had strong IFN-γ secretion (>200 mIU/ml); among 271 naive, 7 (2.58%) and 17 (6.27%) subjects did not show borderline or strong secretion, respectively.ConclusionsIn naive subjects, low IgG titers are relatively long-lasting. Only a third of naive subjects maintain neutralizing responses. After specific stimulation, a very limited number of naive were unable to produce IFN-γ. The results attained in the small group of subjects with breakthrough infection suggest that simultaneous neutralizing antibody titers <20, binding antibody levels/ml <200, and IFN-γ <1,000 mIU/ml in subjects older than 58 may identify at-risk groups.

A Novel Method to Reduce ELISA Serial Dilution Assay Workload Applied to SARS-CoV-2 and Seasonal HCoVs.

Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method using Bayesian hierarchical modelling to reduce the workload of these assays and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from 3 spread-out dilutions correlated with those inferred from 8 consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

Carbon dots hybrid for dual fluorescent detection of microRNA-21 integrated bioimaging of MCF-7 using a microfluidic platform

BackgroundMicroRNAs have short sequences of 20 ~ 25-nucleotides which are similar among family members and play crucial regulatory roles in numerous biological processes, such as in cell development, metabolism, proliferation, differentiation, and apoptosis.ResultsWe reported a strategy for the construction of a dual-emission fluorescent sensor using carbon dots (CDs) and confirmed their applications for ratiometric microRNA-21 sensing and bioimaging of cancer cells in a microfluidic device. The composition of blue CDs (B-CDs) and yellow CDs (Y-CDs) depicts dual-emission behavior which is centered at 409 and 543 nm under an excitation wavelength of 360 nm. With increasing microRNA-21 concentration, the robust and specific binding of DNA probe functionalized B-CDs to complementary microRNA-21 target induced perturbations of probe structure and led to changing fluorescence intensity in both wavelengths. Consequently, the ratio of turn-on signal to turn-off signal is greatly altered. With monitoring of the inherent ratiometric fluorescence variation (ΔF540nm/ΔF410nm), as-prepared BY-CDs were established as an efficient platform for ratiometric fluorescent microRNA-21 sensing, with a wide linear range of 0.15 fM to 2.46 pM and a detection limit of 50 aM.ConclusionsFurthermore, the proposed assay was applied for detecting microRNA-21 in dilute human serum samples with satisfactory recovery and also in MCF-7 cell lines in the range 3000 to 45,000 (cell mL−1) with a detection limit (3 cells in 10 μL), demonstrating the potential of the assay for clinic diagnosis of microRNA-associated disease. More importantly, the images revealed that MCF-7 cells well labeled with BY-CDs could exhibit the applicability of the proposed microfluidic system as an effective cell trapping device in bioimaging.Graphical

Evaluation of the effect of maternally derived antibody on response to MMR vaccine in Thai infants

BackgroundAlthough the number of measles cases declined globally in response to anti-measles immunisation campaigns, measles has re-emerged. A review of current vaccination policies is required to improve measles elimination strategies. MethodsA pseudotype-based virus neutralisation assay (PVNA) was used to measure neutralising antibody titres in serum samples collected from Thai infants at six timepoints before and after two-doses of MMR (1&2) vaccination (ClinicalTrials.gov no. NCT02408926). Vesicular stomatitis virus (VSV) luciferase pseudotypes bearing the haemaglutinin (H) and fusion (F) glycoproteins of measles virus (MeV) were prepared. Serial dilutions of serum samples were incubated with VSV (MeV) pseudotypes and plated onto HEK293-human SLAM1 cells; the neutralising antibody titre was defined as the dilution resulting in 90% reduction in luciferase activity. ResultsNeutralising antibody titres in infants born with high levels of maternal immunity (H group) persisted at the time of the first MMR vaccination, and those infants did not respond effectively by developing protective titres. In contrast, infants with lower maternal immunity (L group) developed protective titres of antibody following vaccination. Responses to the second MMR vaccination were significantly higher (P = 0.0171, Wilcoxon signed-rank test) in the H group. The observed correlation between anti-MeV IgG level and neutralising antibody titre in Thai infants indicates the possibility of using rapid IgG testing as a surrogate measure for neutralising activity to define clinical protection levels within populations. ConclusionThese results demonstrate that varying the timing of the first MMR immunisation according to the level of acquired maternal immunity could increase vaccination immunogenicity and hence accelerate measles eradication.

Target-driven assembly of DNAzyme probes for simultaneous electrochemical detection of multiplex microRNAs.

In this work, we employed target-driven assembly of a Mg2+-dependent DNAzyme to develop an ultrasensitive electrochemical biosensor for the simultaneous detection of miRNA-21 and miRNA-141. The target miRNAs could hybridize with two partial DNAzymes, facilitating the formation of a stable and active Mg2+-dependent DNAzyme. With the help of the Mg2+ cofactor, the DNAzyme could circularly cleave the ferrocene (Fc) or methylene blue (MB) labelled hairpin probes and release Fc and MB labels from the electrode surface, which could significantly amplify the current suppression to achieve multiple detection of small amounts of miRNA-21 and miRNA-141. This electrochemical biosensor showed high sensitivity and selectivity for the simultaneous detection of miRNA-21 and miRNA-141. Furthermore, the proposed method was also successfully applied for the determination of miRNA-21 and miRNA-141 from diluted serum samples. Overall, the proposed sensor showed several considerable advantages including simple preparation, high sensitivity, and enzyme-free signal amplification. Therefore, the proposed electrochemical biosensor could be used as a highly efficient amplification strategy for simultaneous detection of various miRNA biomarkers in bioanalysis and clinical diagnostics.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Ratiometric Detection of microRNA Using Hybridization Chain Reaction and Fluorogenic Silver Nanoclusters.

miRNA (miR)-155 is a potential biomarker for breast cancers. We aimed at developing a nanosensor for miR-155 detection by integrating hybridization chain reaction (HCR) and silver nanoclusters (AgNCs). HCR serves as an enzyme-free and isothermal amplification method, whereas AgNCs provide a built-in fluorogenic detection probe that could simplify the downstream analysis. The two components were integrated by adding a nucleation sequence of AgNCs to the hairpin of HCR. The working principle was based on the influence of microenvironment towards the hosted AgNCs, whereby unfolding of hairpin upon HCR has manipulated the distance between the hosted AgNCs and cytosine-rich toehold region of hairpin. As such, the dominant emission of AgNCs changed from red to yellow in the absence and presence of miR-155, enabling a ratiometric measurement of miR with high sensitivity. The limit of detection (LOD) of our HCR-AgNCs nanosensor is 1.13 fM in buffered solution. We have also tested the assay in diluted serum samples, with comparable LOD of 1.58 fM obtained. This shows the great promise of our HCR-AgNCs nanosensor for clinical application.

Enhanced Plasmonic Biosensor Utilizing Paired Antibody and Label-Free Fe3O4 Nanoparticles for Highly Sensitive and Selective Detection of Parkinson’s α-Synuclein in Serum

Parkinson’s disease (PD) is an acute and progressive neurodegenerative disorder, and diagnosis of the disease at its earliest stage is of paramount importance to improve the life expectancy of patients. -Synuclein (-syn) is a potential biomarker for the early diagnosis of PD, and there is a great need to develop a biosensing platform that precisely detects -syn in human body fluids. Herein, we developed a surface plasmon resonance (SPR) biosensor based on the label-free iron oxide nanoparticles (Fe3O4 NPs) and paired antibody for the highly sensitive and selective detection of -syn in serum samples. The sensitivity of the SPR platform is enhanced significantly by directly depositing Fe3O4 NPs on the Au surface at a high density to increase the decay length of the evanescent field on the Au film. Moreover, the utilization of rabbit-type monoclonal antibody (-syn-RmAb) immobilized on Au films allows the SPR platform to have a high affinity-selectivity binding performance compared to mouse-type monoclonal antibodies as a common bioreceptor for capturing -syn molecules. As a result, the current platform has a detection limit of /, which is 20,000-fold lower than that of commercial ELISA. The improved sensor chip can also be easily regenerated to repeat the -syn measurement with the same sensitivity. Furthermore, the SPR sensor was applied to the direct analysis of -syn in serum samples. By using a format of paired -syn-RmAb, the SPR sensor provides a recovery rate in the range from 94.5% to 104.3% to detect the -syn in diluted serum samples precisely. This work demonstrates a highly sensitive and selective quantification approach to detect -syn in human biofluids and paves the way for the future development in the early diagnosis of PD.

Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) for Highly Sensitive Detection of Single-Stranded Nucleic Acids and Proteins.

Development of versatile sensing methods for sensitive and specific detection of clinically relevant nucleic acids and proteins is of great value for disease monitoring and diagnosis. In this work, we propose a novel isothermal Self-primer EXPonential Amplification Reaction (SPEXPAR) strategy based on a rationally engineered structure-switchable Metastable Hairpin template (MH-template). The MH-template initially keeps inactive with its self-primer overhanging a part of target recognition region to inhibit polymerization. The present targets can specifically compel the MH-template to transform into an "activate" conformation that primes a target-recyclable EXPAR. The method is simple and sensitive, can accurately and facilely detect long-chain single-stranded nucleic acids or proteins without the need of exogenous primer probes, and has a high amplification efficiency theoretically more than 2n. For a proof-of-concept demonstration, the SPEXPAR method was used to sensitively detect the characteristic sequence of the typical swine fever virus (CSFV) RNA and thrombin, as nucleic acid and protein models, with limits of detection down to 43 aM and 39 fM, respectively, and even the CSFV RNA in attenuated vaccine samples and thrombin in diluted serum samples. The SPEXPAR method may serve as a powerful technique for the biological research of single-stranded nucleic acids and proteins.

Two doses of the SARS-CoV-2 BNT162b2 vaccine enhance antibody responses to variants in individuals with prior SARS-CoV-2 infection.

Understanding the impact of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the response to vaccination is a priority for responding to the coronavirus disease 2019 (COVID-19) pandemic. In particular, it is necessary to understand how prior infection plus vaccination can modulate immune responses against variants of concern. To address this, we sampled 20 individuals with and 25 individuals without confirmed previous SARS-CoV-2 infection from a large cohort of health care workers followed serologically since April 2020. All 45 individuals had received two doses of the Pfizer-BioNTech BNT162b2 vaccine with a delayed booster at 10 weeks. Absolute and neutralizing antibody titers against wild-type SARS-CoV-2 and variants were measured using enzyme immunoassays and pseudotype neutralization assays. We observed antibody reactivity against lineage A, B.1.351, and P.1 variants with increasing antigenic exposure, through either vaccination or natural infection. This improvement was further confirmed in neutralization assays using fixed dilutions of serum samples. The impact of antigenic exposure was more evident in enzyme immunoassays measuring SARS-CoV-2 spike protein–specific IgG antibody concentrations. Our data show that multiple exposures to SARS-CoV-2 spike protein in the context of a delayed booster expand the neutralizing breadth of the antibody response to neutralization-resistant SARS-CoV-2 variants. This suggests that additional vaccine boosts may be beneficial in improving immune responses against future SARS-CoV-2 variants of concern.

Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene

Single-nucleotide polymorphism (SNP) is a typical way of genetic variation and commonly used as genetic marker to indicate various types of human diseases. We herein report a non-enzymatic and label-free sensing method for accurate and ultrasensitive differentiation of SNP in K-ras gene. The presence of target mutant K-ras gene (MtDNA) leads to the formation of “three-way junctions” (3WJ) structure with an exposure toehold site, which initiates the toehold strand displacement reaction (TSDR) to form multi-arm DNAzyme nanostructures. These DNAzymes further cyclically cleave the hairpin DNA substrates and release many G-rich fragments to interact with organic dye thioflavin T (ThT) and yield significantly enhanced fluorescence for sensitive discrimination of MtDNA. However, the presence of wild K-ras gene (WtDNA) trigger a base pairing frame shift on the 3WJ structure and result in the closure of the toehold site, thus neither TSDR nor DNAzyme-assistant cleavage reaction is initiated, and no obvious fluorescent signal is observed. Importantly, this strategy shows high sensitivity with a detection limit of 3.63 fM and exhibits high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. Moreover, this approach can be used to analyze target MtDNA in diluted serum samples, which demonstrates its potential application for future clinical diagnostics.

A logic dual-channel detection of Hox transcript antisense intergenic RNA using graphene switch and padlock probe-based exponential rolling circle amplification assay

Sensitive and simultaneous detection of multiple low-abundance long noncoding RNA (lncRNA) biomarkers promises an early diagnosis of lung cancer. In this work, we demonstrate a quadruple logic-mode (YES, NOT, AND and OR) sensing platform for dual specific sequences (T1 and T2) of Hox transcript antisense intergenic RNA (HOTAIR) based on padlock probe-based exponential rolling circle amplification (P-ERCA) assay, producing dual-channel electrochemical and fluorescence (FL) signals simultaneously (AND/OR mode). T1/T2 acts as a key unlocking the P-ERCA amplification strategy, combining with fluorescein Cy3/Cy5 and Pb2+/Cd2+ labeled polydopamine nanospheres (PDA-Pb2+/PDA-Cd2+) give out the dual-channel signals, realizing the quantitative detection of T1 and T2. The dual-channel assay exhibits a wide linear range of 1 fM to 100 pM with low detection limits of 0.25 fM and 0.3 fM for these two targets, respectively. Furthermore, the proposed genosensor possesses high sensitivity, selectivity, and anti-interference ability. With standard addition method, our work shows the recovery experimental T1 value of 86–114.6 % and T2 of 82.2–159 % in diluted serum samples. Moreover, the proposed genosensor obtained comparable detection results of HOTAIR content in whole RNA extracted from the real blood samples with those performed by standard qRT-PCR technique. Results in this study confirmed the feasibility of using the HOTAIR genosensing strategy in bioanalysis.

Highly Sensitive Homogeneous Electrochemiluminescence Biosensor for Alkaline Phosphatase Detection Based on Click Chemistry-Triggered Branched Hybridization Chain Reaction.

Alkaline phosphatase (ALP) has been used as a diagnostic index of clinical diseases since its expression level is closely related to many pathological processes. In this work, a highly sensitive electrochemiluminescence (ECL) method for the determination of ALP based on a click chemistry-induced branched hybridization chain reaction (BHCR) for signal amplification and ultrafiltration technology for the separation of homogeneous amplification products is introduced. ALP can release copper ions from a Cu2+/PPi complex by hydrolyzing pyrophosphoric acid, which initiates click chemistry in the system. A BHCR amplification is triggered afterward by the long single-stranded DNA (ssDNA) generated by click chemistry, resulting in a three-dimensional double-stranded DNA (dsDNA) with a large molecular weight. Based on the characteristic that Ru(phen)32+ can stably insert into the groove of dsDNA, a large amount of Ru(phen)32+ is retained together with the amplified product after ultrafiltration, and therefore a significantly enhanced ECL signal can be obtained. The test results show that this method can be used for the quantitative determination of ALP ranging from 0.002 to 50 U/L, with a detection limit of 0.7 mU/L. This method has also been confirmed to have good selectivity and anti-interference, and the results of the analysis of the ALP content in the diluted serum samples are satisfactory, showing great application potential in clinical diagnosis.

Target-triggered autocatalytic sequence recycling for sensitive and simultaneous detection of microRNA and mRNA via multi-donor iFRET signal amplification

Simultaneous monitoring of different biomarker molecules can potentially offer high accuracy for disease diagnosis. In this work, by using a new biological auto-cycling proximity recording (APR) approach and multi-donor-induced fluorescence resonance energy transfer (iFRET), we establish a multiplexed and sensitive fluorescent method for simultaneous detection of microRNA-155 and osteopontin mRNA for accurate alcoholic liver disease diagnosis. The presence of the two target sequences triggers the APR process via two toehold-mediated strand displacement reactions for the generation of many dsDNAs with the ROX and Cy5 dyes linked to their termini. Subsequent excitation of the SYBR Green I dye intercalated into the dsDNA strands exhibits amplified fluorescence at distinct wavelengths via iFRET for sensitive and simultaneous detection of microRNA-155 and osteopontin mRNA. With the synergistic signal amplification by APR and iFRET, our method shows sub-femtomolar detection limits for the two RNA sequences (e.g., 0.5 and 0.3 fM for microRNA-155 and osteopontin mRNA, respectively). In addition, such a method can also realize high selectivity and the detection of the two RNA sequences in diluted serum samples, indicating its potential application for accurate diagnosis of other diseases.

Ultrasensitive electrochemical detection of microRNA based on in-situ catalytic hairpin assembly actuated DNA tetrahedral interfacial probes

Selective and sensitive detection of microRNA is crucial for early diagnosis and pathogenesis of disease. Here, we established a novel electrochemical biosensor for simple and accurate analysis of the tumor biomarker microRNA-141, which was based on in-situ catalytic hairpin assembly (CHA) actuated DNA tetrahedral (DTN) interfacial probes. Two hairpin structures used for CHA reaction were placed on the DTN, in which the hairpin H1 on the one vertex of DTN and hairpin H2 embedded in adjacent edge, respective. The target microRNA-141 could open the hairpin H1 and activated the in-situ CHA reaction between H1 and H2 to alter the conformational of DTN, increasing the chances of the direct interaction between methylene blue (MB) and the electrode surface, leading to an increase in the electrochemical signal. Meanwhile, the released miRNA-141 could unfold another H1, enabling the enzyme-free recycling of the target to obtain amplified electrochemical signals. Moreover, the in-situ catalytic hairpin assembly reaction on DTN could shorten the reaction time and enhance the sensitivity. The established biosensor exhibited a wide linear dynamic range of miRNA-141 from 1 fM to 100 pM with a detection limit of 0.32 fM. Besides, the approach can discriminate the target miRNA from mismatched ones with excellent selectivity and can be successfully applied in diluted serum samples, holding great potential for sensitive detection of various biomarkers clinically.

A subfemtomolar electrochemical DNA biosensor realized by in-situ grafting of gold nanoparticle/neutral red on the terminal of hairpin probe as the signal tag

Hairpin-structured oligonucleotide (HSO) probe presents higher analytical specificity than the traditional linear analog since the analytical signal driven by conformation change of HSO strictly follows the base-pairing principle. However, the homogeneous liquid-based preparation of electroactive HSO is complicated and time/reagent-consuming, and the signal output is limited. Herein, a novel strategy for electroactive HSO synthesis is proposed by direct labeling of gold nanoparticle (GNP) and neutral red (NR) on the terminal of the HSO probe that pre-confined on a gold electrode, which easily realizes the synthesis of the electroactive HSO and fabrication of electrochemical DNA biosensor synchronously. Electrochemical results show that the assembled electroactive tag (NR-GNP) presents excellent electroactivity and stronger electrochemical signal intensitiy than the single-point labeling HSO. The HSO probe after in-situ labeling of electroactive NR-GNP tag remains the intrinsic outstanding hybridization specificity of the pristine one. A good linear relationship between the signal attenuation of the biosensor and the logarithm of target DNA concentration is achieved in seven orders (1.0 fM ~ 1.0 nM). The detection limit is down to subfemtomolar level. When the developed biosensor is applied for target DNA analysis in a 10% (V/V) diluted serum sample, satisfactory recoveries from 93.6 to 101.6%, showing promising values of the strategy in practical clinic diagnosis.

Integrated dielectrophoretic and impedimetric biosensor provides a template for universal biomarker sensing in clinical samples.

The detection and quantification of nucleic acid and proteomic biomarkers in bodily fluids is a critical part of many medical screening and diagnoses. However, majority of the current detection platforms are not ideal for routine, rapid, and low-cost testing in point-of-care settings. To address this issue, we developed a concept for a disposable universal point-of-care biosensor that can detect and quantify nucleic acid and proteomic biomarkers in diluted serum samples. The central tenet of sensing is the use of dielectrophoresis, electrothermal effects, and thermophoresis to selectively and rapidly isolate the biomarkers of interest in electrodes and then quantify using electrical impedance. When the sensor was applied to quantify microRNA and antigen biomarker molecules directly in diluted serum samples, it produced a LOD values in the fM range and sensitivity values from 1012 to 1015 Ω/M with a 30 min assay time and assay cost of less than $50 per assay.

VALIDATION OF CHEMILUMINESCENT ASSAY FOR CANINE THYROID STIMULATING HORMONE IN RED PANDAS (AILURUS FULGENS FULGENS).

Thyroid abnormalities have been anecdotally reported in red pandas (Ailurus fulgens fulgens); however, definitive diagnosis is hampered by a lack of established reference ranges and validated diagnostic tests. The chemiluminescent assay for canine thyroid stimulating hormone (cTSH) has been validated for use in domestic canids and felids. This study aims to validate the cTSH assay for use in red pandas. Validation was performed via serial dilutions of banked serum samples (n = 15) and both inter- and intra-assay testing. High estimated recoveries and low coefficients of variability indicate that the cTSH assay accurately and consistently measures TSH concentrations in red panda serum. Further studies to generate red panda age and sex TSH reference ranges are indicated.

Convenient and highly sensitive electrochemical biosensor for monitoring acid phosphatase activity

Acid phosphatase (ACP) is a potential biomarker for different diseases. Using the DNA extension-based biological signal amplification and redox recycling of electrochemical species on reduced graphene oxide (rGO)-modified electrode, we established here a highly sensitive, label-free and electrode immobilization-free electrochemical assay for ACP in diluted human serum samples. Target analyte of ACP catalyzes the de-phosphorylation of the 3′-PO4 termini of a ssDNA into 3′−OH on the magnetic beads, which subsequently initiates strand extension into long guanine-rich ssDNAs by the terminal deoxynucleotidyl transferase with pre-defined ratio of dGTP to dATP. The magnetic accumulation of the beads on the rGO-modified electrode thus yield largely enhanced current signals, due to redox recycling oxidation of the many guanine bases in the extended DNAs mediated by [Ru(bpy)3]2+. Such significant current amplification can therefore lead to sensitive electrochemical detection of ACP in the range from 5 to 100 U/L with a low detection limit of 0.52 U/L. High selectivity of the sensing method can also be obtained and the monitoring of ACP in diluted serum samples is demonstrated. With the significant advantages of simplicity and sensitivity, this assay approach holds great potential for convenient detection of other biomolecules such as alkaline phosphatase at low levels.

Effective clearance of uremic toxins using functionalised silicon Nanoporous membranes.

In-house fabricated silicon nanoporous membranes (SNMs), functionalized for efficient clearance of uremic toxins, can lead to compact and portable dialysis systems. Efficacy of 15nm thick SNMs, with average pore diameter of 8nm, was tested for dialysis of two uremic toxins - urea and creatinine using custom made teflon apparatus of 2, 10 and 30ml. The apparatus consisted of two reservoirs, with the cis containing the uremic fluid, and the trans containing the dialysate. Peristalsis was found to enhance the clearance rate by a factor of four as compared to unstirred condition. Functionalisation of the SNMs reduced protein binding, and surface binding of urea from 23% to negligible values. A lateral array of nine SNMs and a new design for the dialysis apparatus, increased the clearance rate by a factor of twelve from that of the single SNM. The arrays cleared about 42% of urea and 48% of creatinine from 30ml of diluted serum samples, in 15min. Periodic replacement of the trans fluid cleared about 81% of high concentration uremic toxins from the cis reservoir in 45 mins. The SNM arrays are stable, reproducible, and with the superior clearance rates for urea and creatinine, they have the potential to be used as membranes for portable hemodialysers.