Dual-mode colorimetric and fluorescence detection of BRCA1 based on a CRISPR-Cas12a system.

Breast cancer, the most common malignant tumor in the world, seriously threatens human life and health. Early diagnosis of breast cancer may help enhance the survival rate. In this work, a colorimetric and fluorescent dual-mode biosensor based on the CRISPR-Cas12a system was constructed to detect the breast cancer biomarker BRCA1. The intact G4 DNA, with the assistance of K+ and hemin, catalyses the oxidation of o-phenylenediamine (OPD) with the assistance of hydrogen peroxide (H2O2), generating the oxidation product 2,3-diaminophenazine (DAP), which has distinct absorption and fluorescence peaks. The presence of the target BRCA1 activates the trans-cleavage activity of CRISPR-Cas12a, leading to the cleavage of G4 DNA and inhibiting the catalytic oxidation of OPD. Target BRCA1 was quantitatively determined by measuring both the absorbance and fluorescence intensity of DAP. The detection limits were calculated to be 0.615 nM for the colorimetric method and 0.289 nM for the fluorescence method. The dual-mode biosensor showed good selectivity and reliability for BRCA1 and can resist interference from complex substrates, and it has great potential in biomedical detection.

DNA tetrahedron-based dual-signal fluorescence detection of apoE4 gene sites on a microplate reader.

As a neurodegenerative disorder, Alzheimer's disease (AD) is characterized by cognitive dysfunction and behavioral impairment. Among the various genetic risk factors for AD, apoE4 gene plays a pivotal role in the onset and progression of AD, and detection of apoE4 gene holds significance for prevention and early diagnosis of AD. Herein, dual-signal fluorescence detection of fragments associated with apoE ε4 allele near codon 112 (Tc1) and codon 158 (Tc2) was achieved using DNA tetrahedron nanostructure (DTN). The Förster resonance energy transfer (FRET) process in the DTN was initiated in which the nucleic acid intercalating dye thiazole orange (TO) served as the donor and the cyanine dyes of cyanine3 (Cy3) and cyanine5 (Cy5) at the two vertices of DTN served as the acceptors. In the presence of Tc1 and Tc2, the FRET process between TO and the cyanine dyes was hindered by the enzymatic cleavage reaction, which ensures the dual-signal fluorescence assay of apoE4 gene sites. The limit of detection for Tc1 and Tc2 was estimated to be 0.82 nM and 0.77 nM, respectively, and the whole assay was accomplished within 1 h on a microplate reader. The proposed method thus possesses the advantages of easy operation, short detection time, and high-throughput capability.

Construction of a Dual-Functional, Reversible, Ratiometric, Golgi-Targeting Fluorescent Probe for Real-time Monitoring of Dynamics of Intracellular Redox Homeostasis.

Intracellular redox homeostasis is highly important for the physiological processes of living organisms. Real-time monitoring of the dynamics of this intracellular redox process is pivotal but challenging because the biological redox reactions involved in the process are reversible and require at least one pair of oxidizing and reducing species. Thus, biosensors for investigating intracellular redox homeostasis need to be dual-functional, reversible, and, ideally, ratiometric in order for them to have real-time monitoring capacity and to provide accurate imaging information. In light of the importance of the redox pair between ClO- and GSH in living organisms, herein, we used the phenoselenazine (PSeZ) moiety as an electron donor and a reaction site to design a coumarin-based fluorescent probe, PSeZ-Cou-Golgi. After successive treatment with ClO- and GSH, the probe PSeZ-Cou-Golgi experienced an oxidation of selenium (Se) to selenoxide (Se═O) by ClO- and a subsequent reduction of Se═O to Se by GSH. The redox reactions alternatively changed the electron-donating strength of the donor in the probe PSeZ-Cou-Golgi, in turn affecting the intramolecular charge transfer process that resulted in the reversible, ratiometric change of fluorescence from red to green. After four cycles of reversible ClO-/GSH detection during in vitro experiments, the probe PSeZ-Cou-Golgi still had good performance. With the Golgi-targeting group, the probe PSeZ-Cou-Golgi was able to monitor the dynamic change of the ClO-/GSH-mediated redox state during Golgi oxidative stress, making it a versatile molecular tool. More importantly, the probe PSeZ-Cou-Golgi could facilitate the imaging of the dynamic redox state during acute lung injury progression.

Surface plasmon resonance detection of UV irradiation-induced DNA damage and photoenzymatic repair processes through specific interaction between consensus double-stranded DNA and p53 protein.

DNA damage, such as DNA lesions and strand breaks, impairs normal cell functions and failure in the DNA repair process could lead to gene mutation, cell apoptosis and disease occurrence. p53 is a tumor suppressor and DNA-binding protein, and DNA damage might affect their interaction and the subsequent p53 function. Herein, real-time monitoring of DNA damage and repair processes through DNA-p53 protein interaction was performed by surface plasmon resonance (SPR). The target DNA with consecutive pyrimidine nucleobases was first damaged upon UVC (254 nm) irradiation and then photoenzymatically repaired under UVA (365 nm) irradiation. The as-formed double-stranded (ds) DNA between probe DNA and normal, damaged or repaired target DNA was immobilized on the sensor chips, followed by the injection of p53 protein. By measuring the SPR signals under different cases, the DNA damage and repair processes could be conveniently monitored. The SPR signals were inversely proportional to the UVC doses ranging from 0.021 to 1.26 kJ m-2, providing a viable means for the quantification of the DNA damage level. The binding affinity between p53 and the dsDNA formed upon the hybridization of probe DNA and normal, damaged, or photoenzymatically repaired target DNA was estimated. This is the first report on measuring the equilibrium dissociation constant (KD) between the p53 protein and the dsDNA with photodamaged or repaired target sequences. The sensing strategy by SPR thus opens a new avenue for real-time measurement of the DNA damage and the repair processes.

Ultraviolet C Irradiation-Induced Dehybridization of Double-Stranded Oligonucleotides: Mechanism Investigation and Label-Free Measurement of the Photodamage Level.

Elucidating the mechanism and estimating the extent of conformation change of double-stranded DNA (dsDNA) upon ultraviolet (UV) exposure are of vital importance for understanding the DNA photodamage process. The existing research was mainly focused on the lesions of single-stranded DNA (ssDNA) and involved off-site measurement of the photodamage level. In this work, short-wavelength UV (UVC) (254 nm) irradiation was demonstrated to induce the dehybridization of dsDNA due to the loss of paring capacity of photodamaged pyrimidine nucleobases. The intrinsic programmability of dsDNA enabled researchers to rationally design the on-demand dehybridization sites. The spatial conformation switch of dsDNA caused by UVC irradiation could be evolved into a label-free sensing platform for the on-site measurement of the DNA photodamage level.

Populations of Puccinia striiformis f. sp. tritici in Winter Spore Production Regions Spread from Southwestern Oversummering Areas in China.

Stripe rust, caused by Puccinia striifomis f. sp. tritici (Pst), is one of the most destructive wheat diseases in China. Understanding the interregional dispersal of Pst inoculum is important for controlling the disease. In the present study, wheat stripe rust samples collected from the winter spore production and oversummering regions in November 2018 to March 2019 were studied through virulence testing and molecular characterization. From 296 isolates, 96 races were identified using a set of 19 Chinese wheat cultivars and 111 races were identified using 18 Yr single-gene lines as differentials. The isolates from Hubei province in the winter spore production area had the highest similarity in virulence with those from eastern Yunnan in the oversummering area. Molecular characterization using 13 simple-sequence repeat and 43 Kompetitive allele specific PCR-single nucleotide polymorphism markers supported the conclusion that the Pst populations in the winter spore production regions were from Guizhou and eastern Yunnan, key oversummering areas in the southwest. Furthermore, an analysis of wind movement at the 700-hPa high altitude also supported the conclusion of spore dispersal from the southwestern oversummering region to the south-central winter spore production region. The results of this study provide an epidemiological basis for deploying various effective resistance genes in different regions to control stripe rust.

Ratiometric electrochemical detection of miRNA based on DNA nanomachines and strand displacement reaction.

MicroRNAs (miRNAs) play an important role in regulating gene expression in cells. Abnormal expression of miRNAs has been associated with a variety of diseases. A ratiometric electrochemical method for miRNA detection based on DNA nanomachines and strand displacement reaction was developed. Signal probe with ferrocene label and reference probe with methylene blue label were immobilized on gold nanoparticle (AuNP)-coated magnetic microbeads (AuNP-MMBs). The miRNA triggers the strand displacement reaction and forms a duplex with the protect probe, releasing one end of the DNA walker (DW); the released DW hybridizes with the ferrocene (Fc)-labeled signal probe. The signal probe detached from AuNP-MMBs upon cleavage of the Nb.BbvCI enzyme. The oxidation peak of MB moieties on the reference probe remains unchanged and the signals of Fc moieties on the signal probe are inversely proportional to the concentrations of miRNA. The ratio between Fc moieties at 0.35 V and MB moieties at -0.22 V (vs. Ag/AgCl) was used to quantify the expression level of miRNA with a detection limit down to 0.12 fM. The ratiometric assay possesses a strong ability to eliminate interference from environmental changes, thus offering the high selectivity of miRNA from the complexed biosystems, holding great significance for miRNA sensing. A ratiometric assay with high selectivity of miRNA has been developed based on DNA nanomachines and strand displacement reaction.

Inhibition of TNFR1 Attenuates LPS Induced Apoptosis and Inflammation in Human Nucleus Pulposus Cells by Regulating the NF-KB and MAPK Signalling Pathway.

Intervertebral disc degeneration (IDD) is accompanied by nucleus pulposus (NP) cell apoptosis, inflammation, and extracellular matrix degradation. Tumour necrosis factor receptor 1 (TNFR1) is a receptor of TNF-α, and is deeply involved in the processes of IDD. However, the effect of TNFR1 inhibition on IDD is not clear. Herein, we report that TNFR1 was increased in LPS-treated HNPCs. The aim of this study was to investigate the potential therapeutic effect of TNFR1 siRNA and selective antagonists of TNFR1 (GSK1995057) on HNPC damage. The results showed that the blockade of TNFR1 by TNFR1 siRNA and GSK1995057 effectively suppressed the cell viability loss, apoptosis, and inflammation induced by LPS in HNPCs. Furthermore, we found that TNFR1 siRNA and GSK1995057 inhibited activation of the NF-KB and MAPK signalling pathways in LPS-stimulated HNPCs. In summary, the blockade of TNFR1 effectively suppressed LPS-induced apoptosis and inflammation in HNPCs through the NF-KB and MAPK signalling pathways. This revealed that the blockade of TNFR1 may provide a potential therapeutic treatment for IDD.

Electrochemical determination of caspase-3 using signal amplification by HeLa cells modified with silver nanoparticles.

An electrochemical sensor capable of quantitative determination of caspase-3 activities was developed. A thiolated peptide whose sequence contained a caspase-3 cleaved site and a cell penetration sequence was preimmobilized onto an electrode. The quantification of caspase-3 was accomplished after cell penetration and the subsequent adsorption of silver nanoparticles (AgNPs). The oxidation current of AgNPs was found to be inversely proportional to the concentration of caspase-3 between 0.02 and 0.2 U/mL. A detection limit of 0.02 U/mL for caspase-3 was achieved due to the large number of positively charged AgNPs adsorbed onto the negatively charged cells. The proof of concept was demonstrated by monitoring the cleavage of surface-confined peptide substrates by caspase-3 in cell lysates. The current sensor could be extended to detect cells by replacing the surface-confined peptide with aptamers that recognize cells. Thus, the use of a cell as a matrix for AgNPs shows excellent potential for constructing electrochemical sensors and provides a useful alternative for sensor development in the future. Cells modified with silver nanoparticles were utilized as the electrochemical readout of an electrochemical assay.

Nanomaterials for Modulating the Aggregation of ß-Amyloid Peptides.

The aberrant aggregation of amyloid-ß (Aß) peptides in the brain has been recognized as the major hallmark of Alzheimer's disease (AD). Thus, the inhibition and dissociation of Aß aggregation are believed to be effective therapeutic strategiesforthe prevention and treatment of AD. When integrated with traditional agents and biomolecules, nanomaterials can overcome their intrinsic shortcomings and boost their efficiency via synergistic effects. This article provides an overview of recent efforts to utilize nanomaterials with superior properties to propose effective platforms for AD treatment. The underlying mechanismsthat are involved in modulating Aß aggregation are discussed. The summary of nanomaterials-based modulation of Aß aggregation may help researchers to understand the critical roles in therapeutic agents and provide new insight into the exploration of more promising anti-amyloid agents and tactics in AD theranostics.

Dissecting the Effect of Salt for More Sensitive Label-Free Colorimetric Detection of DNA Using Gold Nanoparticles.

Taking advantage of the protection effect of single-stranded DNA oligonucleotides, gold nanoparticles (AuNPs) remain dispersed and retain a red color with the addition of a low concentration of salt, while AuNPs would aggregate in the presence of double-stranded DNA. This difference has been used to design label-free colorimetric sensors for DNA detection. NaCl is the most commonly used salt to induce the aggregation of AuNPs. In this work, we aimed to test if other salts can provide even better sensor performance and to understand the effects of the cations and anions in salts. We first studied the effect of anions, including halides (NaF, NaCl, NaBr, and NaI), and other common salts (NaNO3, NaClO4, Na2SO4, Na2S2O3, sodium phosphate, and sodium citrate). Among them, weakly adsorbing ones such as F-, citrate, and phosphate appeared to yield better sensitivity than Cl-. Anions can directly adsorb on the AuNPs and affect DNA adsorption. We then tested cations, and only group 1A metals (LiCl, NaCl, KCl, RbCl, and CsCl) can signal DNA adsorption, while divalent metals (MgCl2, CaCl2, MnCl2, and NiCl2) barely showed the effect of DNA. CsCl only works for strongly adsorbing DNA, such as A15, but not weakly adsorbing T15. Overall, NaF is a better salt than NaCl by having a 2.3-fold higher sensitivity, which was confirmed in a DNA sensing assay. This work has identified a better salt yielding higher sensitivity, and sensing work relying on the change of the aggregation state of AuNPs can benefit from this study.

Three-Dimensional DNA Nanomachine Combined with Toehold-Mediated Strand Displacement Reaction for Sensitive Electrochemical Detection of MiRNA.

MicroRNA (miRNA) serves as an ideal biomarker for diagnosis, prognosis, and therapy of various human cancers. The rationally designed three-dimensional (3D) DNA nanomachine was constructed on the matrixes of magnetic beads, and the high density of gold nanoparticles (AuNPs) on each magnetic bead and further enlargement of the AuNPs lead to the anchoring of numerous DNA walkers and signal probes on the AuNPs. With the combination of toehold-mediated strand displacement reaction (SDR), amplified electrochemical detection of miRNA is performed. The existence of miRNA triggers the toehold-mediated SDR and the released DNA walker probe is hybridized with the ferrocene (Fc)-tagged signal probe. The cleavage of the duplex by the nicking endonuclease detaches the signal probe from the magnetic nanocomposites. The oxidation current of Fc moieties was found to be inversely proportional to the concentrations of miRNA-182 between 1.0 fM and 2 pM. The assay is highly selective for discrimination of miRNAs with similar sequences. The feasibility of the method for sensitive detection of the expression levels of miRNA-182 from serum samples of glioma patients at different stages was demonstrated. The sensing protocol holds great promise for early diagnosis and prognosis of the cancer cases with abnormal miRNA expression.

Sensitive and selective monitoring of the DNA damage-induced intracellular p21 protein and unraveling the role of the p21 protein in DNA repair and cell apoptosis by surface plasmon resonance.

The cyclin-dependent kinase inhibitor p21 protein is a critical regulator that mediates various biological activities, such as cell cycle progression, apoptosis, and cellular senescence. As a DNA damage-inducing agent, doxorubicin could reactivate the transcriptional activity of p53 and modulate the p21 protein level. In this work, sensitive and selective monitoring of the intracellular p21 protein in doxorubicin-treated breast cancer cells was conducted using surface plasmon resonance (SPR). The fluidic channels were pre-immobilized with double stranded (ds) DNA/proliferating cell nuclear antigen (PCNA) for the capture of the p21 protein. The incorporation of the anti-p21 antibody-streptavidin conjugate pre-formed between streptavidin and biotinylated anti-p21 antibody that specifically recognizes the p21 protein leads to signal amplification. The detection limit of 0.85 pM for the p21 protein was lower than that using the commercial enzyme-linked immunosorbent assay (ELISA) kit. The treatment of MCF-7 breast cancer cells with wild-type p53 by various doses of doxorubicin leads to differences in the extent of DNA damage. Low-level DNA damage by low-dose doxorubicin up-regulates the p21 level, and p21 exerts its anti-apoptotic function, causing p53-dependent cell cycle arrest and DNA repair. However, massive DNA damage by high-dose doxorubicin represses the expression of the p21 protein through increased proteasome activity, leading to cell apoptosis. The proposed method is sensitive, selective and label-free, holding great promise for the assay of the DNA damage-induced intracellular p21 protein and understanding of p21 protein-mediated cell cycle arrest, DNA repair, and cell apoptosis.

NIR Light-Responsive Hollow Porous Gold Nanospheres for Controllable Pressure-Based Sensing and Photothermal Therapy of Cancer Cells.

Pressure-based signal transduction has attracted recent and extensive attention due to its high sensitivity and simplicity. The most popular way to generate gas pressure relies on catalyst-mediated decomposition of H2O2. Despite its high sensitivity, this method lacks spatial and temporal control of the reaction, and may suffer from variations due to the dead time of mixing. In this work, we report a new reaction using near-infrared (NIR) light to heat hollow porous gold nanospheres (AuNSs) for thermal decomposition of NH4HCO3. Comparisons were made on these two systems especially on controlled pressure production. As an example of application, our light-controlled system was used for the detection of MCF-7 cancer cells by attaching the S2.2 aptamer on the AuNSs. The detection limit was as low as 2 cells/mL. Meanwhile, the heat produced by the AuNSs was used to induce localized hyperthermia at the surface of the cancer cells. This interesting theranostic system provides new insights into pressure-based sensing and may inspire new analytical applications.

Hyaluronic Acid-Modified Porous Carbon-Coated Fe3O4 Nanoparticles for Magnetic Resonance Imaging-Guided Photothermal/Chemotherapy of Tumors.

Chemotherapy is an effective method for treating cancer, clinically. However, side effects of drug and multidrug resistance restrict its application. In recent years, the combined treatment of chemotherapy and photothermal therapy (PTT) is becoming a promising method for treating cancer. PTT utilizes nanomaterials absorbing near-infrared light and producing heat to acquire advanced hyperthermia strategy for cancer treatment. Carbon nanomaterials with good biocompatibility, high surface area, and excellent photothermal properties are an excellent nanoplatform for drug delivery and PTT. Herein, porous carbon-coated magnetite nanoparticles (PCCMNs) were successfully synthesized by a one-pot solvothermal method. Magnetite, a contrast agent, can be used for magnetic resonance imaging. Hyaluronic acid was used to modify the PCCMNs to achieve targeted therapy. The obtained nanohybrid with a good photothermal effect can realize combined PTT/chemotherapy and will be a promising nanoplatform for high efficacy theranostics.

Real-time surface plasmon resonance monitoring of site-specific phosphorylation of p53 protein and its interaction with MDM2 protein.

Phosphorylation serves as an important post-translational modification implicated in cellular signaling and regulation. In this work, real-time monitoring of site-specific phosphorylation of p53 protein by several protein kinases, followed by its interaction with MDM2 protein was conducted using surface plasmon resonance (SPR). The binding of phosphorylated p53 to MDM2 yields a smaller SPR signal in comparison with that in the case of unphosphorylated p53 protein. Three specific protein kinases were involved in the in situ phosphorylation of the surface-confined p53 protein, and the binding kinetics between the phosphorylated p53 and MDM2 protein was monitored. The results indicate that phosphorylation of Ser15 and Ser37 at the p53 transactivation domain 1 (TAD1) by DNA-dependent protein kinase (DNA-PK) is critical for inhibiting the p53-MDM2 interaction, and the weaker binding affinity is most likely caused by the hydrophobicity change in the vicinity of the MDM2-binding motif or phosphorylation-induced p53 conformational change. In contrast, phosphorylation of Ser46 at the p53 TAD2 domain by c-Jun NH2-terminal kinase 2α2 (JNK2α2) exerts a weaker influence on the binding affinity, whereas phosphorylation of Ser376 and Ser378 at the C-terminus of p53 by protein kinase C (PKC) appears to have little effect. The feasibility of the method for the screening of the DNA-PK inhibitor and the inhibitor of p53-MDM2 interaction has been demonstrated and the half-maximal inhibitory concentration (IC50) values of wortmannin and Nutlin-3 (21 nM and 83 nM, respectively) were highly comparable with those obtained by other methods. The proposed method holds great promise for monitoring protein phosphorylation and unraveling the post-translational modification mechanism.

Dual-channel surface plasmon resonance monitoring of intracellular levels of the p53-MDM2 complex and caspase-3 induced by MDM2 antagonist Nutlin-3.

MDM2 can mediate the degradation of tumor suppressor p53 through an autoregulatory feedback loop, in which MDM2 abolishes wild-type p53 function and accelerates malignant transformation. However, the incorporation of MDM2 antagonist Nutlin-3 could reactivate the transcriptional activity of p53, up-regulate caspase-3, and induce apoptosis. In this work, the simultaneous and label-free monitoring of p53-MDM2 complex and caspase-3 levels in cancer cells before and after Nutlin-3 treatment was conducted using dual-channel surface plasmon resonance (SPR). The p53-MDM2 complex was captured in one fluidic channel covered with consensus double-stranded (ds)-DNA, while the other channel was pre-immobilized with caspase-3-specific biotinylated DEVD-containing peptides. To amplify the SPR signals, the attachment of streptavidin (SA)-conjugated anti-MDM2 antibody in both channels was achieved. The signal diversity before and after Nutlin-3 treatment is indicative of the difference in the levels of the intracellular p53-MDM2 complex and caspase-3. The limit of detection for p53-MDM2 and caspase-3 down to 4.54 pM and 0.03 ng mL-1, respectively, was attained. Upon treatment with Nutlin-3, MCF-7 cancer cells with wild-type p53 showed decreased expression of the p53-MDM2 complex and an increased caspase-3 level, while MDA-MB-231 cancer cells with mutant p53 exhibited an elevated caspase-3 level and unchanged p53-MDM2 complex expression. The apoptosis of MCF-7 and MDA-MB-231 cancer cells upon Nutlin-3 treatment follows a p53-dependent and a p53-independent pathway, respectively. The proposed method is sensitive, selective and label-free, holding great promise for assaying intracellular p53-MDM2 complex and caspase-3 levels and differentiating Nutlin-3-mediated p53-dependent or p53-independent apoptotic pathways.

A porous carbon nitride modified with cobalt phosphide as an efficient visible-light harvesting nanocomposite for photoelectrochemical enzymatic sensing of glucose.

A porous carbon nitride (PCN) modified with cobalt phosphides (CoP) was synthesized. In this nanocomposite, the CoP (in different weight fractions) serves (a) as the electron acceptor to accelerate the photoinduced charge separation, and (b) as the photosensitizer to increase the photoelectrochemical (PEC) response to visible light. Dissolved oxygen acts as the electron acceptor to generate PEC current. If glucose oxidase (GOx) catalyzes the oxidation of glucose, dissolved oxygen is consumed. This leads to the suppression of photocurrent. The photocathode biosensor has a linear response in the 0.05 to 0.7 mM glucose concentration range and a 1.1 µM limit of detection. Graphical abstractSchematic of a photoelectrochemical glucose biosensor based on the use of cobalt phosphide-modified porous carbon nitrides. PCN: porous carbon nitride; CoP: cobalt phosphide.

Sensitive and simultaneous surface plasmon resonance detection of free and p53-bound MDM2 proteins from human sarcomas.

Murine double minute 2 (MDM2) is an oncoprotein mediating the degradation of the tumor suppressor p53 protein. The physiological levels of MDM2 protein are closely related to malignant transformation and tumor growth. In this work, the simultaneous and label-free determination of free and p53-bound MDM2 proteins from sarcoma tissue extracts was conducted using a dual-channel surface plasmon resonance (SPR) instrument. Free MDM2 protein was measured in one fluidic channel covered with the consensus double-stranded (ds)-DNA/p53 conjugate, while MDM2 bound to p53 was captured by the consensus ds-DNA immobilized onto the other channel. To achieve higher sensitivity and to confirm specificity, an MDM2-specific monoclonal antibody (2A10) was used to recognize both the free and p53-bound MDM2 proteins. The resultant method afforded a detection limit of 0.55 pM of MDM2. The amenability of the method to the analysis of free and p53-bound MDM2 proteins was demonstrated for normal and sarcoma tissue extracts from three patients. Our data reveal that both free and total MDM2 (free and bound forms combined) proteins from sarcoma tissue extracts are of much higher concentrations than those from normal tissue extracts and the p53-bound MDM2 protein only constitutes a small fraction of the total MDM2 concentration. In comparison with enzyme-linked immunosorbent assay (ELISA), the proposed method possesses higher sensitivity, is more cost-effective, and is capable of determining free and p53-bound MDM2 proteins in clinical samples.

Voltammetric determination of the Alzheimer's disease-related ApoE 4 gene from unamplified genomic DNA extracts by ferrocene-capped gold nanoparticles.

A sensitive method is described for detection of the apoE 4 gene detection which is important for early diagnosis of Alzheimer's disease. It is based on signal amplification by using ferrocene (Fc) capped gold nanoparticles modified with streptavidin. The immobilized oligonucleotide probe captures complementary apoE 4 gene. This is followed by the specific recognition of the GCGC sequences which are hydrolyzed by the restriction enzyme HhaI. Cleavage only occurs at the complementary apoE 4 duplex, while mismatches prevent enzymatic cleavage. Thus, the apoE 4 sequence can be discriminated against other apoE sequences. Benefitting from amplified signal by Fc-capped nanoparticle/streptavidin and the recognition of HhaI, the detection limit is as low as 0.1 pM of the ApoE 4 gene. Four genomic DNA samples extracted from blood were analyzed for the presence of the apoE 4 gene. The approach presented here will provide viable proof-of-principle for an enzyme-assisted electrochemical assay for the apoE 4 gene in genomic DNAs. Graphical abstract Schematic presentation of amplified voltammetric detection of Alzheimer's Disease-related apoE 4 gene from unamplified genomic DNA extracts via ferrocene capped gold nanoparticle/streptavidin.