Assessing base-resolution DNA mechanics on the genome scale.

Intrinsic DNA properties including bending play a crucial role in diverse biological systems. A recent advance in a high-throughput technology called loop-seq makes it possible to determine the bendability of hundred thousand 50-bp DNA duplexes in one experiment. However, it's still challenging to assess base-resolution sequence bendability in large genomes such as human, which requires thousands of such experiments. Here, we introduce 'BendNet'-a deep neural network to predict the intrinsic DNA bending at base-resolution by using loop-seq results in yeast as training data. BendNet can predict the DNA bendability of any given sequence from different species with high accuracy. To explore the utility of BendNet, we applied it to the human genome and observed DNA bendability is associated with chromatin features and disease risk regions involving transcription/enhancer regulation, DNA replication, transcription factor binding and extrachromosomal circular DNA generation. These findings expand our understanding on DNA mechanics and its association with transcription regulation in mammals. Lastly, we built a comprehensive resource of genomic DNA bendability profiles for 307 species by applying BendNet, and provided an online tool to assess the bendability of user-specified DNA sequences (http://www.dnabendnet.com/).

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.

Overview of the Design and Application of Photothermal Immunoassays.

Developing powerful immunoassays for sensitive and real-time detection of targets has always been a challenging task. Due to their advantages of direct readout, controllable sensing, and low background interference, photothermal immunoassays have become a type of new technology that can be used for various applications such as disease diagnosis, environmental monitoring, and food safety. By modification with antibodies, photothermal materials can induce temperature changes by converting light energy into heat, thereby reporting specific target recognition events. This article reviews the design and application of photothermal immunoassays based on different photothermal materials, including noble metal nanomaterials, carbon-based nanomaterials, two-dimensional nanomaterials, metal oxide and sulfide nanomaterials, Prussian blue nanoparticles, small organic molecules, polymers, etc. It pays special attention to the role of photothermal materials and the working principle of various immunoassays. Additionally, the challenges and prospects for future development of photothermal immunoassays are briefly discussed.

Colorimetric Immunoassays with Boronic Acid-Decorated, Peroxidase-like Metal-Organic Frameworks as the Carriers of Antibodies and Enzymes.

The sensitivity of immunoassays is generally limited by the low signal reporter/recognition element ratio. Nanomaterials serving as the carriers can enhance the loading number of signal reporters, thus improving the detection sensitivity. However, the general immobilization strategies, including direct physical adsorption and covalent coupling, may cause the random orientation and conformational change in proteins, partially or completely suppressing the enzymatic activity and the molecular recognition ability. In this work, we proposed a strategy to load recognition elements of antibodies and enzyme labels using boronic acid-modified metal-organic frameworks (MOFs) as the nanocarriers for signal amplification. The conjugation strategy was proposed based on the boronate ester interactions between the carbohydrate moieties in antibodies and enzymes and the boronic acid moieties on MOFs. Both enzymes and MOFs could catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2, therefore achieving dual signal amplification. To indicate the feasibility and sensitivity of the strategy, colorimetric immunoassays of prostate specific antigen (PSA) were performed with boronic acid-modified Cu-MOFs as peroxidase mimics to catalyze TMB oxidation and nanocarriers to load antibody and enzyme (horseradish peroxidase, HRP). According to the change in the absorbance intensity of the oxidized TMB (oxTMB), PSA at the concentration range of 1~250 pg/mL could be readily determined. In addition, this work presented a site-specific and oriented conjugation strategy for the modification of nanolabels with recognition elements and signal reporters, which should be valuable for the design of novel biosensors with high sensitivity and selectivity.

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.

Background-Quenched Aggregation-Induced Emission through Electrostatic Interactions for the Detection of Poly(ADP-ribose) Polymerase-1 Activity.

Poly(ADP-ribose) polymerase-1 (PARP1) is a potential biomarker and therapeutic target for cancers that can catalyze the poly-ADP-ribosylation of nicotinamide adenine dinucleotide (NAD+) onto the acceptor proteins to form long poly(ADP-ribose) (PAR) polymers. Through integration with aggregation-induced emission (AIE), a background-quenched strategy for the detection of PARP1 activity was designed. In the absence of PARP1, the background signal caused by the electrostatic interactions between quencher-labeled PARP1-specitic DNA and tetraphenylethene-substituted pyridinium salt (TPE-Py, a positively charged AIE fluorogen) was low due to the fluorescence resonance energy transfer effect. After poly-ADP-ribosylation, the TPE-Py fluorogens were recruited by the negatively charged PAR polymers to form larger aggregates through electrostatic interactions, thus enhancing the emission. The detection limit of this method for PARP1 detection was found to be 0.006 U with a linear range of 0.01~2 U. The strategy was used to evaluate the inhibition efficiency of inhibitors and the activity of PARP1 in breast cancer cells with satisfactory results, thus showing great potential for clinical diagnostic and therapeutic monitoring.

Overview on the Development of Alkaline-Phosphatase-Linked Optical Immunoassays.

The drive to achieve ultrasensitive target detection with exceptional efficiency and accuracy requires the advancement of immunoassays. Optical immunoassays have demonstrated significant potential in clinical diagnosis, food safety, environmental protection, and other fields. Through the innovative and feasible combination of enzyme catalysis and optical immunoassays, notable progress has been made in enhancing analytical performances. Among the kinds of reporter enzymes, alkaline phosphatase (ALP) stands out due to its high catalytic activity, elevated turnover number, and broad substrate specificity, rendering it an excellent candidate for the development of various immunoassays. This review provides a systematic evaluation of the advancements in optical immunoassays by employing ALP as the signal label, encompassing fluorescence, colorimetry, chemiluminescence, and surface-enhanced Raman scattering. Particular emphasis is placed on the fundamental signal amplification strategies employed in ALP-linked immunoassays. Furthermore, this work briefly discusses the proposed solutions and challenges that need to be addressed to further enhance the performances of ALP-linked immunoassays.

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.

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.

Liraglutide ameliorates diabetes-associated cognitive dysfunction via rescuing autophagic flux.

The incidence of diabetes-associated cognitive dysfunction is increasing. However, few clinical interventions are available to prevent the disorder. Several researches have shown that liraglutide, as a glucagon-like peptide-1 analog, has protective effects on various neurodegenerative diseases, but its roles in diabetic cognitive dysfunction are rarely reported. This study aims to investigate the protective effects of liraglutide on diabetic cognitive dysfunction and its underlying mechanisms. In vivo, the effects of liraglutide treatment were investigated in a mouse model of type 2 diabetes mellitus (T2DM). In vitro, we investigated the effects of liraglutide on the high-glucose-induced rat primary neurons. The results showed that liraglutide reduced the escape latency and increased the time in effective area in the Morris water maze test, improved the damage of hippocampal and synaptic ultrastructure, and decreased the accumulation of amyloid ß protein in hippocampus of T2DM mice. Furthermore, liraglutide increased the ratio of microtubule-associated protein light 1 chain Ⅱ/Ⅰ, the expression of Beclin1 protein and Lysosome-associated membrane protein 2 in vivo and vitro. Additionally, Bafilomycin A1 which can inhibit the fusion of autophagosome and lysosome partially abolished the effects of liraglutide. These findings indicate liraglutide ameliorates diabetes-associated cognitive dysfunction by rescuing autophagic flux.

Construction of graphene quantum dots-decorated EGFR cell membrane chromatography for screening active components from Peucedanum praeruptorum Dunn.

A novel stability-enhanced graphene quantum dot (GQD)-decorated epidermal growth factor receptor (EGFR) cell membrane chromatography was constructed to study the potential application of GQDs in bioaffinity chromatography, and to screen active components acting on EGFR from traditional Chinese medicine (TCM). The carboxyl groups on the surface of GQDs reacted with the amino groups of the amino-silica gel (SiO2-NH2) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO2-GQDs). The EGFR cell membrane was further immobilized on the SiO2-GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO2-GQDs-CMSP). In this way, the cell membrane was firmly immobilized on the decorated silica carrier. The life span and stability of the GQD-decorated cell membrane chromatographic (SiO2-GQDs-CMC) column were both enhanced, and the optimal immobilization conditions of the EGFR cell membrane were also determined. This model was then verified by establishing a SiO2-GQDs-CMC online liquid chromatography-ion trap-time-of-flight (LC-IT-TOF) system to screen possible active components in Peucedanum praeruptorum Dunn. As a result, praeruptorin B (Pra-B) was screened out, and its inhibitory effect against EGFR cell growth was evaluated by the cell counting kit-8 (CCK-8) assay. Molecular docking assay was also conducted to further estimate the interaction between Pra-B and EGFR. Overall, this research indicated that GQDs may be a promising nanomaterial to be used in prolonging the life span of the CMC column, and Pra-B could be a potential EGFR inhibitor so as to treat cancer.

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.

Analysis of codon usage patterns in citrus based on coding sequence data.

BACKGROUND: Codon usage is an important determinant of gene expression levels that can help us understand codon biology, evolution and mRNA translation of species. The majority of previous codon usage studies have focused on single species analysis, although few studies have focused on the species within the same genus. In this study, we proposed a multispecies codon usage analysis workflow to reveal the genetic features and correlation in citrus. RESULTS: Our codon usage analysis workflow was based on the GC content, GC plot, and relative synonymous codon usage value of each codon in 8 citrus species. This approach allows for the comparison of codon usage bias of different citrus species. Next, we performed cluster analysis and obtained an overview of the relationship in citrus. However, traditional methods cannot conduct quantitative analysis of the correlation. To further estimate the correlation among the citrus species, we used the frequency profile to construct feature vectors of each species. The Pearson correlation coefficient was used to quantitatively analyze the distance among the citrus species. This result was consistent with the cluster analysis. CONCLUSIONS: Our findings showed that the citrus species are conserved at the genetic level and demonstrated the existing genetic evolutionary relationship in citrus. This work provides new insights into codon biology and the evolution of citrus and other plant species.

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.