Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: From Disc Activation to Multiplex Detection.

Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnosis of AMI is extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I, and C-creative protein) but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis.

DNA-Redox Cation Interaction Improves the Sensitivity of an Electrochemical Immunosensor for Protein Detection.

A simple DNA-redox cation interaction enhancement strategy has been developed to improve the sensitivity of electrochemical immunosensors for protein detection. Instead of labeling with fluorophores or redox-active groups, the detection antibodies were tethered with DNA single strands. Based on the electrostatic interaction between redox cations ([Ru(NH3)6](3+)) and negatively charged DNA backbone, enhanced electrochemical signals were obtained. Human chorionic gonadotropin (hCG) detection has been performed as a trial analysis. A linear response range up to the concentration of 25 mIU/mL and a detection limit of 1.25 mIU/mL have been achieved, both are comparable with the ultrasensitive enzyme-linked immunosorbent assay (ELISA) tests. The method also shows great selectivity towards hCG over other hormones such as thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH). By and large, our approach bears the merits of cost effectiveness and simplicity of instrumentation in comparison with conventional optical detection methods.

DVD technology-based molecular diagnosis platform: quantitative pregnancy test on a disc.

A diagnosis platform based entirely on DVD technology was developed for on-site quantitation of molecular analytes of interest, e.g., human chorionic gonadotropin (hCG) in urine samples ("quantitative pregnancy test on a disc"). An hCG-specific monoclonal antibody-binding assay prepared on a regular DVD-R was labeled with nanogold-streptavidin conjugates for signal enhancement with a customized silver-staining protocol. An unmodified, conventional computer optical drive was used for assay reading, and free disc-quality analysis software for data processing. The performance (sensitivity and selectivity) of this DVD assay is comparable to that of well-established colorimetric methods (determination of optical darkness ratios) and standard enzyme-linked immunosorbent assays (ELISA). As validated by examining its linear correlation with the ELISA results on the same set of samples, the DVD assay promises to be a low-cost, multiplex, point-of-care (POC) diagnostic tool for physicians and even for individuals at home, producing prompt results.

Functional DNA switches: rational design and electrochemical signaling.

Recent developments in nanoscience research have demonstrated that DNA switches (rationally designed DNA nanostructures) constitute a class of versatile building blocks for the fabrication and assembly of electronic devices and sensors at the nanoscale. Functional DNA sequences and structures such as aptamers, DNAzymes, G-quadruplexes, and i-motifs can be readily prepared in vitro, and subsequently adapted to an electrochemical platform by coupling with redox reporters. The conformational or conduction switching of such electrode-bound DNA modules in response to an external stimulus can then be monitored by conventional voltammetric measurements. In this review, we describe how we are able to design and examine functional DNA switches, particularly those systems that utilize electrochemical signaling. We also discuss different available options for labeling functional DNA with redox reporters, and comment on the function-oriented signaling pathways.

Adenosine-triggered elimination of methylene blue noncovalently bound to immobilized functional dsDNA-aptamer constructs.

Immobilization and electrochemical characterization of specially designed functional DNA-aptamer constructs are of great importance for the development of versatile biosensors (not limited to gene analysis) and the investigation of molecular interactions between DNA and other molecules. We have constructed a "DNA conformational switch" by incorporating the antiadenosine aptamer sequence in the middle of an otherwise cDNA double helix, as its structural change responds to the presence of small molecule ligands (e.g., adenosine). In particular, methylene blue (MB) was used as a model system to probe the rather complex interaction modes between small redox molecules and the dsDNA-aptamer construct. Besides intercalating with the double-stranded DNA stem, MB can stack with a single guanine base in the relatively unstructured aptamer domain or electrostatically bind to the DNA backbone. The decreased surface density of MB after adenosine binding indicated that the ligand-gated structural change of the dsDNA-aptamer construct can eliminate MB molecules that were originally bound to the aptamer domain but not those in the complementary stem.

Molecular and immuno-characteristics of immunoglobulin-like glycoproteins in cancer cell-expressed biomarker, CA215.

RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5-18 days), but unstable at extreme pH's (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo.

CA215 and GnRH receptor as targets for cancer therapy.

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.

Widespread expressions of immunoglobulin superfamily proteins in cancer cells.

RP215 monoclonal antibody (Mab) was shown to recognize a carbohydrate-associated epitope of cancer cell-expressed glycoproteins, known as CA215. Extensive MALDI-TOF MS analysis was performed to search for the molecular identity of CA215. Besides immunoglobulin (Ig) heavy chains, homology to human T-cell receptors (TCR) and Ig-like cell adhesion molecules was also detected. By using RT-PCR and cDNA sequencing, it was observed that as many as 80% of cancer cell lines showed significant levels of gene expressions of TCR-α and TCR-ß. Selected Ig-like cell adhesion molecules such as CD47, CD54, CD58 and CD 147 were also highly expressed among all the cell lines tested. In contrast, co-receptors and co-stimulators of TCR such as CD3, CD4 and CD8 were rarely expressed demonstrating the non-functional nature of TCR in cancer cells. Results of immunohistochemical staining and Western blot assays of cancer cell lines as well as cancerous tissue sections were consistent with these observations. Anti-TCR and anti-human IgG antibodies were shown to induce complement-dependent cytotoxicity and apoptosis of cultured cancer cells indicating the surface nature of Ig-like proteins. Based on these experimental observations, it was hypothesized that the expressions of these immunoglobulin superfamily (IgSF) proteins may be relevant to the immune protection and proliferations of cancer cells during carcinogenesis or cancer progression. Surface-bound TCR-like proteins as well as immunoglobulins may be the potential targets for RP215-based anti-cancer drugs.

Carbohydrate-associated immunodominant epitope(s) of CA215.

RP215 monoclonal antibody (Mab) was initially generated against OC-3-VGH ovarian cancer cells and was shown to react with a cancer-associated carbohydrate epitope in glycoproteins designated as CA215. Additional five high affinity Mabs, designated as RCA-10, -100, -104, -110 and -111, respectively, were generated by using affinity-purified CA215 as the immunogen in this study. All RCA Mabs were found to recognize periodate-sensitive carbohydrate-associated epitope(s) and to pair with RP215 in typical sandwich enzyme immunoassays for the quantification of CA215. When compared with those of RP215, the amino acid sequence homology of the Fab regions ranged from 100% for RCA-100 to 65% for RCA-110, based on which 3 distinct Mab groups were categorized. In vitro TUNEL apoptosis and complement-dependent cytotoxicity assays were performed with these Mabs and found to have comparable inhibitory efficacy to cancer cells. Results of biochemical and immunological assays revealed that RP215, RCA-100 and RCA-10 react with the linear carbohydrate-associated epitope, whereas the others recognize the conformational form of the epitope in CA215. This study has suggested that the unique carbohydrate-associated epitope(s) is immunodominant in mice when immunized with CA215. It remains to be demonstrated if the differential anti-cancer efficacy exists among the distinct groups of these anti-CA215 Mabs.

Positive identification of CA215 pan cancer biomarker from serum specimens of cancer patients.

BACKGROUND: To evaluate the clinical utility of CA215 as a pan cancer biomarker, serum levels of CA215 were determined with clinically defined serum specimens from over 500 cancer patients and compared with results obtained by other nine established cancer markers. The molecular nature of this cancer-associated antigen from selected patients' sera was determined. METHODS: By using improved immunoassays, serum levels of CA215 and other known biomarkers were determined for respective positive detection rates. The molecular size of CA215 from cancer patients was determined by Western blot assay. RESULTS: By using 0.1 AU/ml as the normal cut-off value, the positive rates of CA215 for different cancers were shown to be 52% (lung), 74% (liver), 44% (colon), 61% (esophagus), 60% (stomach), 59% (ovary), 40% (prostate), 71% (breast), 38% (kidney), 41% (pancreas), 51% (cervix), and 83% (lymphoma), respectively. Other cancer markers including AFP, CEA, CA125, CA19-9, CA15-3, Cyfra21-1, Ferritin, beta(2) microglobulin and PSA were also parallelly compared. A combination of CA215 with other tissue-associated cancer markers generally resulted in much higher cancer detection rates. CA215 detected from cancer patients was confirmed to be human immunoglobulins that contain common RP215-specific carbohydrate-associated epitope. CONCLUSION: Through clinical evaluations of serum specimens of various cancer patients, CA215 was confirmed to be human cancer cell-derived immunoglobulins. CA215 is apparently comparable to or better than other known biomarkers for the positive detection and monitoring of many types of human cancers.

Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies.

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH's (or=12) or following NaIO(4) treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Cancer cell expressions of immunoglobulin heavy chains with unique carbohydrate-associated biomarker.

OC-3-VGH ovarian cancer cell line and numerous others from different human tissue origins were studied for their respective expressions of immunoglobulins as well as carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody. With no exceptions, all the cancer cell lines studied so far express human immunoglobulin G (IgG) heavy chains when determined by Western blot or nested RT-PCR with appropriate primers in the constant region. By Western blot assay, it was also shown that greater than 90% of cancer cell lines expressed RP215-specific epitope(s) on the detected heavy chain molecules. Further studies with OC-3-VGH cancer cells revealed the expressions of all immunoglobulin classes, subclasses, heavy as well as light chains. The primary structure of the IgG heavy chains expressed by single cloned cells of this cancer cell line was elucidated. It was shown to be homologous to that of normal human IgG1 heavy chain derived from B cells, except with high content of serine/threonine residues in the variable region. Expressions of other immunoglobulin-related genes were also detected. Widespread expressions of immunoglobulin heavy chains among cancer cells as well as the frequent presence of unique carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody might have important biological implications during carcinogenesis and applications in immunodiagnostics and antibody-based anti-cancer drug developments.

Immobilized DNA switches as electronic sensors for picomolar detection of plasma proteins.

The sensing principle of a new class of DNA conformational switches (deoxyribosensors) is based on the incorporation of an aptamer as the receptor, whose altered conformation upon analyte binding switches on the conductivity of an adjacent helical conduction path, leading to an increase in the measured electrical signal through the sensor. We report herein the rational design and biochemical testing of candidate deoxyribosensors for the detection and quantitation of a plasma protein, thrombin, followed by surface immobilization of the optimized sensor and its electrochemical testing in both a near-physiological buffer solution and in diluted blood serum. The very high detection sensitivity (in the picomolar range) and specificity, as well as the adaptability of deoxyribosensors for the detection of diverse molecular analytes both small and macromolecular, make this novel sensing methodology an extremely promising one. Such synthetic and robust DNA-based electronic sensors should find broad application in the rapid, miniaturized, and automated on-chip detection of many biomedically relevant substances (such as metabolites, toxins, and disease and tumor markers) as well as of environmental contaminants.

Aptamer-based biosensors for label-free voltammetric detection of lysozyme.

This paper reports a simple electrochemical approach for the detection of the ubiquitous protein lysozyme using aptamer-modified electrodes. Anti-lysozyme DNA aptamers were immobilized on gold surfaces by means of self-assembly, for which the surface density of aptamers was determined by cyclic voltammetric (CV) studies of redox cations (e.g., [Ru(NH3)6]3+) bound to the surface via electrostatic interaction with the DNA phosphate backbone. Upon incubation of the electrode with a solution containing lysozyme, the CV response of surface-bound [Ru(NH3)6]3+ changed substantially, and the relative decrease in the integrated charge of the reduction peak can be tabulated as a quantitative measure of the protein concentration. It is significant that the on-chip protein/aptamer binding constant and the optimized surface density to achieve the best detection limit can be evaluated. This biosensor is label-free and offers an alternative, sensitive, and versatile method for protein detection, which is beneficial to the ever-growing interests of fabricating portable bioanalytical devices with simple electrical readout protocols.

Allosterically activated Diels-Alder catalysis by a ribozyme.

We describe the allosteric control of Diels-Alder reactions by a small organic effector, theophylline. This is achieved by converting a Diels-Alder ribozyme into an allosterically regulated system. In contrast to other published systems, we have a bond-forming reaction with two small-molecule substrates and multiple turnover. This system could be very attractive for the development of assays for a variety of analytes and can be regarded as a prototype of fully synthetic signaling cascades.

In vivo measurement of oxygen-derived free radicals during reperfusion injury.

By use of an optimized cytochrome c-based biosensor, superoxide radical production was measured continuously in vivo. The aim of this study was the online detection of superoxide concentration during reperfusion after a variable time of ischemia. Measurements were performed by placing the detecting sensor into gastrocnemius muscle tissue. Ischemia was induced by clamping the vena and arteria femoralis. Current response of the sensor was recorded continuously as an equivalent for superoxide concentration. Ischemia times varied from 5 to 120 minutes. The minimum ischemia time to record superoxide production was 10 minutes. By inducing longer periods of ischemia, an increase in superoxide concentration reached its highest levels at 2 hours. Furthermore, the total time of superoxide production after reperfusion depended on the total time of ischemia.

Bioelectrocatalysis by redox enzymes at modified electrodes.

Self-assembled monolayers of thiolated compounds are used as promoters for protein-electrode reactions. They provide an anchor group based on thiol chemisorptions and also a functional group for effective interaction with the protein. These interactions are often governed by electrostatic attraction. For example, for positively charged proteins, such as cytochrome c and the selenoprotein glutathione peroxidase, mercaptoalkanoic acids have been used. Clay modification of the electrode surface has been found to facilitate the heterogeneous electron transfer process for heme proteins, e.g. cytochrome c, cytochrome P450 and myoglobin. Interestingly, nucleic acids at carbon electrodes and thiol-modified double stranded oligonucleotides act as promoters of the redox communication to proteins, whereas the mechanism is still subject to controversy interpretations. By interacting the protein immobilised at the electrode with species in solution, signal chains have been constructed. The interaction can result in a simple co-ordination or redox reaction, depending on the nature of the reaction partners. For analytical purposes, e.g. biosensors, the electrochemical redox conversion of the immobilised protein is evaluated.