Upconversion luminescence assay for the detection of the vascular endothelial growth factor,a biomarker for breast cancer

Upconversion nanoparticles (UCNPs) of the type α-NaYF₄:Yb³⁺, Er³⁺ and a typical diameter of 6–7 nm were synthesized by thermal decomposition of the respective rare-earth stearate. The oleic acid on the surface of the UCNPs was then replaced by aptamer DNA. The assay was performed in a microplate format with a capture probe immobilized in the wells. Following binding of the vascular endothelial growth factor (VEGF), an auxiliary probe DNA is added that is labeled with UCNPs and binds to the VEGF-loaded capture probe. The method enables highly sensitive and highly specific detection of the VEGF which is a marker for breast cancer. Under the optimum conditions, the intensity of the upconversion luminescence (at excitation/emission wavelengths of 980/541 nm) is linearly proportional to the VEGF concentration in the 50 pM to 2000 pM concentration range, with a 6 pM detection limit. The method was applied to the determination of VEGF in spiked serum, typically at a 500 pM level, and gave recoveries that ranged from 98 to 113 %, with RSDs between 2.9 and 3.6 %. This makes it a viable tool for early diagnosis of breast cancer.

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Graphical abstract An upconversion luminescence based assay is presented for trace analysis of vascular endothelial growth factor. It is based on a sequence fragment-linked technique of target-induced aptamer (AP) and upconversion nanoparticles (UCNPs) and has potential application in the early diagnosis of breast cancer.

     

Fluorescence aggregation assay for the protein biomarker mucin 1 using carbon dot-labeled antibodies and aptamers

Microchimica Acta - We have developed a sensitive fluorescence assay for the protein biomarker mucin 1 (MUC1). It is based on the aggregation of functionalized carbon dots (CDs) in the presence of...     

The mitogen-activated protein kinase phosphatase-1 (MKP-1) gene is a potential methylation biomarker for malignancy of breast cancer

The mitogen-activated protein kinase (MAPK) phosphatase- 1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P<0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P<0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver- operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P<0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.     

Detection of a biomarker for Alzheimer's disease from synthetic and clinical samples using a nanoscale optical biosensor

A nanoscale optical biosensor based on localized surface plasmon resonance (LSPR) spectroscopy has been developed to monitor the interaction between the antigen, amyloid-beta derived diffusible ligands (ADDLs), and specific anti-ADDL antibodies. Using the sandwich assay format, this nanosensor provides quantitative binding information for both antigen and second antibody detection that permits the determination of ADDL concentration and offers the unique analysis of the aggregation mechanisms of this putative Alzheimer's disease pathogen at physiologically relevant monomer concentrations. Monitoring the LSPR-induced shifts from both ADDLs and a second polyclonal anti-ADDL antibody as a function of ADDL concentration reveals two ADDL epitopes that have binding constants to the specific anti-ADDL antibodies of 7.3 x 10(12) M(-1) and 9.5 x 10(8) M(-1). The analysis of human brain extract and cerebrospinal fluid samples from control and Alzheimer's disease patients reveals that the LSPR nanosensor provides new information relevant to the understanding and possible diagnosis of Alzheimer's disease.     

Visualizing hidden components of envelopes non-parametrically in magnetic resonance spectroscopy: Phosphocholine,a breast cancer biomarker

A key advantage of rational polynomials as a quotient of two polynomials is their automatically built-in polar representation. Rational polynomials are thus the most suitable for describing functions with peaks, such as those in magnetic resonance spectra (MRS). The Padé approximant is the most important of these rational polynomials because of its uniqueness for the power series expansion of the given function. Non-parametric analysis through the fast Padé transform (FPT) is a convenient initial step for processing MRS time signals, since it can be carried out once the expansion coefficients of the polynomials are generated from the time signal, without polynomial rooting. We applied the FPT to synthesized MRS time signals similar to those encoded in vitro from breast cancer. Padé-based non-parametric envelopes generated with and without spectra partitioning are studied. Comparisons of these total shape spectra with the related Padé component spectra were made. Phosphocholine (PC) and phosphoethanolamine (PE), separated by a mere 0.001 parts per million of chemical shift, were resolved in the non-parametric partitioned envelopes. However, in the non-parametric FPT without partitioning, a single composite smooth Lorentzian peak (PC \(+\) PE) was generated in envelopes, with no indication whatsoever that two resonances were present. Subsequent parametric analysis (quantification) by the FPT confirmed that PC completely underlies the much more abundant PE. This problem, chosen to illustrate the usefulness in the non-parametric partitioned envelopes, has clinical implications. Namely, PC is a cancer biomarker which thus far was not identified with in vivo MRS using envelopes from the conventional Fourier-based (single-polynomial) processing.     

Porous silicon surfaces: a candidate substrate for reverse protein arrays in cancer biomarker detection

This paper introduces a new substrate for reverse-phase protein microarray applications based on macroporous silicon. A key feature of the microarray substrate is the vastly surface enlarging properties of the porous silicon, which simultaneously offers highly confined microarray spots. The proof of principle of the reverse array concept was demonstrated in the detection of different levels of cyclin E, a possible cancer biomarker candidate which regulates G1-S transition and correlates with poor prognosis in different types of human cancers. The substrate properties were studied performing analysis of total cyclin E expression in human colon cancer cell lines Hct116 and SW480. The absence of unspecific binding and good microarray quality was demonstrated. In order to verify the performance of the 3-D textured macroporous surface for complex biological samples, lysates of the human tissue spiked to different levels with cell extract overproducing cyclin E (Hct116) were arrayed on the chip surface. The samples were spotted in a noncontact mode in 100 pL droplets with spots sizes ranged between 50 and 70 mum and spot-to-spot center distances 100 mum, allowing microarray spot densities up to 14 000 spots per cm(2). The different sample types of increasing complexities did not have any impact on the spot intensities recorded and the protein spots showed good homogeneity and reproducibility over the recorded microarrays. The data demonstrate the potential use of macroporous silicon as a substrate for quantitative determination of a cancer biomarker cyclin E in tissue lysates.     

A fluorescence-based assay for nanogram quantification of proteins using a protein binding ligand

Fluorescence emission has been investigated in the context of estimation of proteins at nanogram levels. A Schiff base ligand with donor-acceptor substituents has been utilized as a fluorescent probe. The potency of this ligand is that it possesses the binding sites for both hydrophobic as well as hydrophilic groups in the proteins. The fluorescence emission of the probe was enhanced in the presence of nanogram levels of protein, which clearly signifies that even the least concentration of the protein is sufficient to perturb the environment around the probe. We demonstrate here that the fluorescence characteristic of the probe can be utilized to estimate even nanogram levels (66 ng-1 microgram mL(-1)) of protein. The major limitation of the currently available standard methods is the range of protein estimation, which terminates at microgram level and the interference due to the specificity of the amino acids, which vary from proteins to proteins. This fluorescence emission-based method is free from interference from any type of buffers, ionic strength of the medium and any specific amino acid residue and is a simple, rapid, single-step, sensitive method of estimation which can be applied to different classes of proteins.     

Vascular endothelial growth factor as a biomarker for the early detection of cancer using a whole cell-based biosensor

Vascular endothelial growth factor (VEGF) is a cytokine and endothelial cell (EC) mitogen that has been studied for its role in angiogenesis of malignant tumors. Elevated quantities of VEGF in the serum and plasma of patients have been correlated with the presence of cancer and metastasis. Since VEGF induces hyperpermeability of EC monolayers, this protein can be detected in vitro with a whole cell-based biosensor. This biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to a cellulose triacetate (CTA) membrane of an ion-selective electrode (ISE). Previous studies regarding this biosensor have shown that when the biosensor was exposed to a model toxin, such as histamine, the response of the biosensor served as an indirect measurement of the presence of histamine. Similarly, the biosensor responds to the presence of VEGF, but is much more sensitive because VEGF is known to be 50,000-fold more potent than histamine when inducing EC hyperpermeability. The ISE response increased with increasing VEGF concentration. Since lower concentrations required more exposure time, the detection limit was established as a function of exposure time (2–10 h). The practical applicability of the biosensor was also established with cultured human melanoma cells WM793 (nonmetastatic) and 1205LU (metastatic). The resultant change in the potential values revealed significant production of VEGF from the 1205LU cells. A VEGF ELISA was performed to confirm the VEGF concentration in each sample. The biosensor closely predicted the concentrations determined through the ELISA. These results support the use of a cell-based ISE as a quick screening method for the presence of VEGF.     

The regulation of microRNA in each of cancer stage from two different ethnicities as potential biomarker for breast cancer

miRNA has recently emerged as a potential biomarker for breast cancer. Even though many studies have identified ethnic variation affecting miRNA regulation, the effect of cancer stage within specific ethnicities on miRNA epigenetic remains unclear. The present study is designed to investigate miRNA regulation from two distinct ethnicities in specific cancer stages (non-Hispanic white and non-Hispanic black) using the TCGA dataset. Differentially expressed miRNAs were calculated by using the edgeR package. miRNAs with the highest or lowest log fold Change from each cancer stage were selected as a potential biomarker. miRNA-gene interaction was analyzed by using spearman correlation analysis, CLUEGO, and DIANA-mirpath. The association of biomarker candidates with diagnostic and prognostic performance was assessed using ROC and Kaplan-Meier survival analysis. miRNA-gene interaction analysis revealed the involvement of selected miRNAs in cancer progression. From eleven selected aberrant miRNAs, four of the miRNAs (hsa-mir-495, hsa-mir-592, hsa-mir-6501, and hsa-mir-937) are significantly detrimental to breast cancer diagnosis and prognosis. Hence, our result provides valuable information to explore miRNA’s role in each cancer stage between non-Hispanic white and non-Hispanic black.     

A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues

Pathological detection using immunohistochemistry (IHC) has become an indispensable process in the diagnosis confirmation of various cancers. However, the production of monoclonal antibodies is always very complex, expensive and time-consuming, and the batch differences are significant due to the corporeity and health statuses of animals may be different. In this work, an aptamer-based histochemistry (aptahistochemistry) assay was developed using a DNA aptamer for specific diagnosis of clinical breast cancer tissue sections. This aptahistochemistry assay can specifically distinguish Luminal A breast cancer molecular subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer molecular subtypes, as well as para-carcinoma tissue, mastitis tissue and normal breast tissue. The accuracy of this aptahistochemistry assay for the diagnosis of Luminal A breast cancer was as high as 80%, which showed a great potential for clinical pathological diagnosis applications.     

A displacement assay for the sensing of protein interactions using sugar-tetraphenylethene conjugates

A displacement sensor array based on sugar-substituted tetraphenylethenes with the aggregation-induced emission feature for proteins to perform screening of protein-protein interactions is demonstrated.     

Inkjet-printed gold nanoparticle electrochemical arrays on plastic. Application to immunodetection of a cancer biomarker protein

Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL(-1) in calf serum, sensitivity of 11.4 nA pg(-1) cm(-2), and a linear dynamic range of 20-400 pg mL(-1).     

Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing

The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach.

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Graphical Abstract A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.

     

Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling

Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor’s surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein’s distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL⁻¹ apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1 h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient’s urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.