ProteinChip System Research Articles

Characterization and Proteomic Analyses of Proinflammatory Cytokines in a Mouse Model of Liver Transplant Rejection.

Liver transplantation (LT) is an effective strategy for the treatment of end-stage liver disease, but immune rejection remains a significant detriment to the survival and prognosis of these LT patients. While immune rejection is closely related to cytokines, the cytokines investigated within previous studies have been limited and have not included a systematic analysis of proinflammatory cytokines. In the present study, we used a protein chip system and proteomics to detect and analyze serum proinflammatory cytokines and differentially expressed proteins in liver tissue in a mouse model of liver transplantation. In addition, bioinformatics analysis was employed to analyze the proinflammatory cytokines and differential changes in proteins in response to this procedure. With these analyses, we found that serum contents of GC-CSF, CXCL-1, MCP-5, and CXCL-2 were significantly increased after liver transplantation, while IL-5, IL-10, and IL-17 were significantly decreased. Results from Gene Ontology (GO) and KEGG pathway analyses revealed that the cytokine-cytokine receptor, Th1/Th2 cell differentiation, and JAK-STAT signaling pathway were enriched in a network associated with the activation of immune response. Results from our proteomic analysis of liver tissue samples revealed that 470 proteins are increased and 50 decreased, including Anxa1, Anxa2, Acsl4, Sirpa, S100a8, and S100a9. KEGG pathway analysis indicated that the neutrophil extracellular trap formation, NOD-like receptor signaling pathway, and leukocyte transendothelial migration were all associated with liver transplant rejection in these mice. Bioinformatics analysis results demonstrated that CXCL-1/CXCL-2 and S100a8/S100a9 were the genes most closely related to the functions of neutrophils and the mononuclear phagocyte system. These findings provide new insights into some of the critical factors associated with liver transplant rejection and thus offer new targets for the treatment and prevention of this condition.

Evaluation of the clinical application of multiple tumor marker protein chip in the diagnostic of lung cancer.

The early diagnostic of lung cancer plays an important role in the prognosis of surgical treatment among lung cancer patients. To evaluate the clinical application of multi-tumor markers protein biochip in the diagnosis of lung cancer, 12 tumor markers were detected in patients with different stages of lung cancer. Serum CA125, CA19-9, Ferritin, CA15-3, CA242, CEA, AFP, NSE, PSA, f-PSA, HGH, and β-HGH were assessed in 506 patients, with 224 patients with lung cancer (including 123 cases of adenocarcinoma, 30 squamous cell carcinoma, 54 small-cell carcinoma, and 17 non classification), 159 patients with benign lung disease and 90 healthy people control by the C-12 multiple tumor protein-chip detective system. The positive rate of C-12 (77.23%) in lung cancer was significantly higher than that of benign lung disease (13.84%) and healthy people (9.76%) (P<.01). In lung cancer, the positive rate of CA199, NSE, CEA, CA242, Ferritin, f-PSA, and CA125 were significantly higher than that of benign lung disease and healthy people. In adenocarcinoma, the positive rate of CA125 (73.53%) was significantly higher than that of squamous cell carcinoma (36.67%) and small-cell carcinoma (56.62%). The C-12 multiple tumor protein-chip detective system has acceptable sensitivity in the diagnostic of lung cancer.

Effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion

To explore the effect of icariin on the proteomic expression profile of bone microvascular endothelial cells of human femoral head against steroids-induced lesion using protein chip system in vitro. Bone microvascular endothelial cells (BMECs) in cancellous bone of femoral head were isolated and harvested in vitro.The model of BMECs injury induced by glucocorticoid and icariin preconditioning was established. The apoptosis of BMECs were detected with terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)methods. The proteomic expression profile of BMECs in each group were detected by protein chip system. The apoptosis rates of hydrocortisone groups (46.9%±5.8%, 78.0%±8.5%) were significantly higher than that of normal group(4.8%±1.3%), meanwhile the apoptosis rate of icariin+ hydrocortisone groups(15.2%±7.7%, 44.6%±5.3%)significantly decreased compared with hydrocortisone group (P<0.001). The results of protein chip showed that the expression of granulocyte colony-stimulating factor (G-CSF) in the hydrocortisone group decreased compared with the control group, meanwhile the expression of IL-4 increased. The expression of G-CSF in the icariin+ hydrocortisone groups increased compared with the hydrocortisone group, meanwhile the expression of IL-4 decreased.No appreciable change was found in the expression of apoptotic factors. The therapy effect of icariin for steroid-induced necrosis of femoral head might be related with the protective action to BMECs and the moderating effect of promoting the proteins expression of pro-angiogenesis factor and inhibiting expression of inflammatory factor.

Weak cation magnetic separation technology and MALDI-TOF-MS in screening serum protein markers in primary type I osteoporosis.

We investigated weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of primary type I osteoporosis. We selected 16 postmenopausal women with osteoporosis and nine postmenopausal women as controls to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were obtained from controls and patients. Serum protein was extracted with the WCX protein chip system; protein fingerprints were examined using MALDI-TOF-MS. The preprocessed and model construction data were handled by the ProteinChip system. The diagnostic models were established using a genetic arithmetic model combined with a support vector machine (SVM). The SVM model with the highest Youden index was selected. Combinations with the highest accuracy in distinguishing different groups of data were selected as potential biomarkers. From the two groups of serum proteins, 123 cumulative MS protein peaks were selected. Significant intensity differences in the protein peaks of 16 postmenopausal women with osteoporosis were screened. The difference in Youden index between the four groups of protein peaks showed that the highest peaks had mass-to-charge ratios of 8909.047, 8690.658, 13745.48, and 15114.52. A diagnosis model was established with these four markers as the candidates, and the model specificity and sensitivity were found to be 100%. Two groups of specimens in the SVM results on the scatterplot were distinguishable. We established a diagnosis model, and provided a new serological method for screening and diagnosis of osteoporosis with high sensitivity and specificity.

A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients

BackgroundWe report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.ResultsFive cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.ConclusionThis protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

Analysis of Plk1-Bora interaction using a protein chip system

We investigated the interaction between Polo-Box Domain (PBD) of polo-like kinase 1 (Plk1) and Bora peptide based on protein-protein interactions using the Proteochip (Proteogen, Korea) technology. Plk-1 protein conjugated with GST was bound to anti-GST antibody immobilized on the surface of the Proteochip, after which a Bora peptide labeled with a fluorescent dye, Cy5, was interacted with the Plk-1 protein on the chip in a dose-dependent manner. A chemical compound E8 was screened as a novel inhibitor against the Plk1-Bora interaction using the Proteochip. The compound significantly inhibited NCI-H460 (lung cancer) cell proliferation in vitro in a dose-dependent manner. The protein chip-based Plk1-Bora binding system will serve a novel promising tool which is applicable for screening of novel inhibitors against Plk1, regardless of the enzyme activity.

Serum Hepcidin Levels and Reticulocyte Hemoglobin Concentrations as Indicators of the Iron Status of Peritoneal Dialysis Patients

Hepcidin is the key mediator of renal anemia, and reliable measurement of serum hepcidin levels has been made possible by the ProteinChip system. We therefore investigated the iron status and serum hepcidin levels of peritoneal dialysis (PD) patients who had not received frequent doses of an erythrocytosis-stimulating agent (ESA) and had not received iron therapy. In addition to the usual iron parameters, the iron status of erythrocytes can be determined by measuring reticulocyte hemoglobin (RET-He). The mean serum hepcidin level of the PD patients (n = 52) was 80.7 ng/mL. Their serum hepcidin levels were significantly positively correlated with their serum ferritin levels and transferrin saturation (TSAT) levels, but no correlations were found between their serum hepcidin levels and RET-He levels, thereby suggesting that hepcidin has no effect on the iron dynamics of reticulocytes. Since low serum levels of CRP and IL-6, biomarkers of inflammation, were not correlated with the serum hepcidin levels, there is likely to be a threshold for induction of hepcidin expression by inflammation.

Characterization of Prefibrillar Tau Oligomers in Vitro and in Alzheimer Disease

Neurofibrillary tangles, composed of insoluble aggregates of the microtubule-associated protein Tau, are a pathological hallmark of Alzheimer disease (AD) and other tauopathies. However, recent evidence indicates that neuronal dysfunction precedes the formation of these insoluble fibrillar deposits, suggesting that earlier prefibrillar Tau aggregates may be neurotoxic. To determine the composition of these aggregates, we have employed a photochemical cross-linking technique to examine intermolecular interactions of full-length Tau in vitro. Using this method, we demonstrate that dimerization is an early event in the Tau aggregation process and that these dimers self-associate to form larger oligomeric aggregates. Moreover, using these stabilized Tau aggregates as immunogens, we generated a monoclonal antibody that selectively recognizes Tau dimers and higher order oligomeric aggregates but shows little reactivity to Tau filaments in vitro. Immunostaining indicates that these dimers/oligomers are markedly elevated in AD, appearing in early pathological inclusions such as neuropil threads and pretangle neurons as well as colocalizing with other early markers of Tau pathogenesis. Taken as a whole, the work presented herein demonstrates the existence of alternative Tau aggregates that precede formation of fibrillar Tau pathologies and raises the possibility that these hierarchical oligomeric forms of Tau may contribute to neurodegeneration.

Differentiation ofCampylobacterSpecies by Surface-Enhanced Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry

The genus Campylobacter contains several, widespread pathogens causing food-borne diseases of zoonotic nature in humans. In case of outbreaks, the differentiation of closely related Campylobacter is essential for epidemiological studies, which investigate the routes of geographical spread and ways of transmission. Recent advances in mass spectrometry (MS) have shown that matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS is a valuable tool for speciation of bacteria such as Campylobacter. Surface-enhanced laser desorption/ionization (SELDI)-TOF-MS is a specific MALDI-TOF application that combines a chip-based chromatographic enrichment of proteins with TOF-MS. This pilot study aims at investigating for the first time whether SELDI-TOF-MS can be applied for discrimination of Campylobacter at the level of species and even strains. Campylobacter type-strains and isolates from different regions were cultured and subsequently subjected to physicochemical lysis. Protein lysates were then applied on CM10 and IMAC30 ProteinChip Array surfaces and analyzed using a PCS 4000 SELDI Protein Chip System (Bio-Rad Laboratories). By comparison of the spectra from Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis, and Campylobacter lari, 166 and 160 different protein peaks were observed (p<0.05) using CM10 and IMAC30 chips, respectively. Development of classification trees, comprising 2-4 of these peaks, allows for discrimination of different Campylobacter species and even strains. Moreover, species and strains can be sufficiently separated from each other by hierarchical cluster analysis. Thus, SELDI-TOF-MS is a promising tool to differentiate Campylobacter species and even strains. Species/strain-specific ions observed in addition to well-established markers identified by MALDI-TOF might be of value for future Campylobacter-identifying algorithms. To further clarify the potential advantages of this method, our results have to be validated against several independent test datasets of, preferably, a multitude of prospectively collected different isolates and compared with other typing techniques.

Development of surface plasmon resonance immunosensor for the novel protein immunostimulating factor

The immunostimulating factor (ISTF) plays an important role in immunopathologic changes associated with Actinobacillus actinomycetemcomitans where it is located in the outer cell membrane. We describe a novel method by which ISTF can be monitored using a protein chip system based on surface plasmon resonance (SPR). The affinity of ISTF to its monoclonal antibodies and other proteins was compared. In fabrication of an ISTF immunosensor, a calix[4]crown ether monolayer was anchored onto a gold surface for use as an artificial protein linker system, and then characterized by Fourier Transform infrared reflection absorption spectroscopy, atomic force microscopy, and cyclic voltammetry. Anti-ISTF was immobilized onto the monolayer, and the interaction between ISTF and its antibodies was investigated by SPR. The calix[4]crown ether was found to assemble as a monolayer on the gold surface. Orientation and accessibility of anti-ISTF were assessed by the selective binding of ISTF. Modification of the SPR chip with the calix[4]crown monolayer provides a reliable and simple experimental platform for investigation of isolated proteins under experimental conditions resembling those of their native environment.

CEA is an Independent Prognostic Indicator that is Associated with Reduced Survival and Liver Metastases in SCLC

Small-cell lung cancer (SCLC) is classified into two stages, limited-stage small-cell lung cancer (LS-SCLC) and extensive-stage small-cell lung cancer (ES-SCLC), with ES-SCLC being associated with reduced survival. However, disease stage alone is not a very precise predictor of survival time. The aim of this study is to search for a prognostic indicator that can be used in conjunction with the staging system in SCLC. We performed a prospective analysis of 116 patients with SCLC undergoing standard chemotherapy with or without radiotherapy. Expression levels of 12 tumor markers were measured at the time of diagnosis by a commercial protein chip system, and patients were followed up for a maximum of 54 weeks. CEA was the most frequently detected tumor marker in SCLC, with 32.8% of samples scoring positive, and was also the only marker that correlated with overall survival. The average survival time was 16.78 months for patients with CEA below 5 ng/ml and 11.4 months for patients with CEA above 5 ng/ml (P < 0.001). In contrast, no association was found between tumor stage and overall survival. CEA expression was independent of tumor stage (P = 0.930). CEA is an independent prognostic factor that can be used in conjunction with disease stage in SCLC.

Using SELDI-TOF MS to identify serum biomarkers in NSCLC

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide with poor differential diagnosis of the disease and with low response rates to standard chemotherapy. Individuals classified as Stage I by the present clinical-based scoring system may display survival characteristics ranging from 10 to 90 %. Identification of reliable biomarkers to detect NSCLC at early stages is therefore a subject of extensive research. Surface-enhanced laser-desorption-ionization time-of-flight mass spectrophotometry (SELDI-TOF-MS) combines chromatographic surfaces with solid-state TOF-MS for the high throughput analysis of proteins in diverse clinical samples such as serum, urine, cerebrospinal fluids and has overcome the limitations of earlier proteomic approaches. The method has been widely used for biomarker discovery. Therefore, in this study we used the SELDI-TOF protein Chip system to achieve a stratification based on the protein expression profile and to identify differentially expressed proteins in sera from 50 patients with NSCLC and 50 healthy controls. IMAC 30-Cu protein chips were used to fractionate the proteins according to their metal binding affinity. The arrays were analyzed using the Ciphergen Protein Chip Reader PB IIa. The spectra were normalized to the total ion current of the mass to charge between 3000 — 10 000 Da. Protein peak clustering and classification analysis were performed using the Express 3.0 Biomarker Pattern Analysis Software. Several proteins with masses ranging from 2000-20000 Da were differentially expressed between NSCLC patients and healthy controls. Among the peak clusters revealing differences we selected two specific clusters as prospective candidate biomarkers in sera of the patients. High expression of one peak and underexpression of the other with respect to the healthy control sera were evaluated in a blind test in terms of the ability to classify a separate group of patients and controls.

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser-Asp-Val (SDV) sequence and integrin alpha v beta 3. P11 (HSDVHK) is a novel antagonistic peptide of integrin alpha v beta 3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin-binding site of integrin alpha v beta 3. The Arg-Gly-Asp (RGD)-binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin-vitronectin. P11 showed a strong antagonism against alpha v beta 3-GRGDSP interaction with an IC(50) value of 25.72+/-3.34 nM, whereas the value of GRGDSP peptide was 1968.73+/-444.32 nM. The binding-free energies calculated from the docking simulations for each P11 and RGD peptide were -3.99 and -3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5-fold lower K(i) value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in alpha v beta 3. The analyzed docked poses suggest that a divalent metal-ion coordination was a common driving force for the formation of both SDV/alpha v beta 3 and RGD/alpha v beta 3 complexes. This is the first report on the specific recognition of the RGD-binding site of alpha v beta 3 by a non-RGD containing peptide using a computer-assisted proteomic approach.

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P<0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.

A search for novel markers of alcohol abuse using magnetic beads and MALDI‐TOF/TOF mass spectrometry

Objective markers are required to assess excessive alcohol consumption, which can lead to a various medical and social problems. In this study, we carried out serum peptidome analyses using the ClinProt(™) system, which consists of magnetic beads and MALDI-TOF/TOF MS, to find novel biomarkers of alcohol abuse in 16 chronic alcoholic patients that were hospitalized for a rehabilitation program. A total of 22 peaks were found to be significantly altered during abstinence. Out of these 22 peaks, 3 peaks that had an m/z of 3000 or less and substantial peak intensities were subjected to MS/MS analysis followed by a MASCOT search. The 1466 Da and the 1616 Da peptides were upregulated on admission and were identified as fragments of fibrinopeptide A and phosphorylated fibrinopeptide A, respectively. On the other hand, the 2660 Da peptide, which was downregulated on admission and increased during abstinence, was identified as a fragment of the fibrinogen α C chain. These peaks were not detectable by the SELDI-TOF MS ProteinChip(®) system analysis. The alterations in these peaks induced by alcohol abuse were also seen in γ glutamyltransferase nonresponders. These protein fragments may be additional biomarkers for excessive alcohol drinking.

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N ɛ-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry: serum protein profiling in seminoma patients

Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) allows rapid protein profiling of complex biological mixtures. We analyzed testicular germ cell cancer serum samples to differentiate between cancer and controls with a special focus on beta-hCG-negative seminomas. Proteomic spectra were generated by the ProteinChip system and analyzed by the proteomic platform "proteomic.net". For statistical analysis, an artificial intelligence learning algorithm was used. The classification algorithm correctly identified the pattern in 90.4% of the patients. Decision trees predicted seminomas with 91.5% sensitivity and 89.4% specificity. Seminoma patients with normal beta-hCG serum level were correctly predicted with 80% sensitivity and 70% specificity. Our study demonstrates protein profiles of testicular germ cell cancer patients that differ in a highly significant degree from normal controls. Validation of these findings may enable proteomic profiling to become a valuable tool, especially for aftercare.

SELDI-TOF-MS-based Serum Proteomic Screening in Combination with CT Scan Distinguishes Renal Cell Carcinoma from Benign Renal Tumors and Healthy Persons

In order to identify the proteomic differences between renal cell carcinoma (RCC) and benign renal tumors, we analyzed 168 serum samples from 65 RCC patients, 34 patients with benign renal tumors, and 69 healthy persons using the IMAC-Cu2+ ProteinChip system by surface enhanced laser desorption/ionization mass spectrometry technology. Two decision trees were generated by Biomarker Pattern software to distinguish between RCC versus healthy and RCC versus patients with benign tumors, respectively. Although the sensitivity and specificity of the RCC vs. healthy decision tree were 97.6% and 95.7%, respectively, it could not be used to distinguish RCC from benign renal tumors. The sensitivity of a blind test process using RCC, benign tumors and healthy persons were 92%. The specificity of the test process was 35.3% for benign tumors and 95.5% for healthy persons. The sensitivity and specificity of the RCC-Benign tumors decision tree were 85.7% and 95.5%, respectively. The blind test process using RCC, benign tumors and healthy persons also showed significant results. The sensitivity was 90.0%. The specificity was 95.7% for healthy persons and 90.0% for benign tumors. Combining these data with the results of CT scanning, the sensitivity can be improved over the use of either CT and decision tree analysis and the specificity may reach 100%. Two peaks with molecular masses of 3887.11 Da and 11079.8 Da were detected that are potentially useful for the diagnosis or screening of RCC. It was found that these two peaks can be used, not only to distinguish the RCC vs. healthy cases, but also to distinguish RCC from benign renal tumors. In combination with CT scanning, the sensitivity and specificity of the diagnosis of renal tumors can be improved. However, the decision tree constructed for RCC and healthy persons may not present good specificity for use in distinguishing malignant from benign renal tumors.

Detection and identification of specific serum biomarkers in papillary thyroid cancer

To detect and identify the potential specific serum biomarkers for diagnosis of papillary thyroid cancer. Samples of 35 patients with papillary thyroid carcinoma, 40 patients with benign thyroid nodule and 34 healthy individuals were analyzed using the SELDI-TOF ProteinChip System and bioinfomation technology to find the differential peaks which were separated by HPLC and then further analyzed by LC-MS/MS. The protein sequences were analyzed by SEQUEST software and searched in Bioworks database. The top six mass-to-charge ratio (M/Z) peaks with the smallest P value were 6651, 6452, 7653, 7932, 15 106 and 15 848 Da, respectively. The 6651 and 6452 Da proteins were weakly expressed in papillary thyroid carcinoma but highly expressed in benign thyroid nodules and healthy individuals. The differences had statistical significance (P < 0.01). The 7653, 7932, 15 106, 15 848 Da proteins were highly expressed in papillary thyroid carcinoma but weakly expressed in benign thyroid nodules and healthy individuals. The differences were statistically significant (P < 0.01). Combination of these six proteins, using the method of leave-one-out to make crossing detection, the specificity of discriminating papillary thyroid carcinoma and non-cancer was 88.0%, and its sensitivity was 92.5%. The 6651 and 6452 Da proteins were identified as apolipoprotein C-I and apolipoprotein C-III, respectively. The 7653 and 15 106 Da proteins were identified as the same protein-alpha-globin, and the 7932 and 15,848 Da proteins were identified as the same protein-beta-globin. The detection of differentially expressed apolipoprotein C-I, apolipoprotein C-III, alpha-globin, and beta-globin may have utility for diagnosis of papillary thyroid carcinoma and are worthy of further investigation.

Integrated three-dimensional optical MEMS for chip-based fluorescence detection

This paper presents a novel fluorescence sensing chip for parallel protein microarray detection in the context of a 3-in-1 protein chip system. This portable microchip consists of a monolithic integration of CMOS-based avalanche photo diodes (APDs) combined with a polymer micro-lens, a set of three-dimensional (3D) inclined mirrors for separating adjacent light signals and a low-noise transformer-free dc–dc boost mini-circuit to power the APDs (ripple below 1.28 mV, 0–5 V input, 142 V and 12 mA output). We fabricated our APDs using the planar CMOS process so as to facilitate the post-CMOS integration of optical MEMS components such as the lenses. The APD arrays were arranged in unique circular patterns appropriate for detecting the specific fluorescently labelled protein spots in our study. The array-type APDs were designed so as to compensate for any alignment error as detected by a positional error signal algorithm. The condenser lens was used as a structure for light collection to enhance the fluorescent signals by about 25%. This element also helped to reduce the light loss due to surface absorption. We fabricated an inclined mirror to separate two adjacent fluorescent signals from different specimens. Excitation using evanescent waves helped reduce the interference of the excitation light source. This approach also reduced the number of required optical lenses and minimized the complexity of the structural design. We achieved detection floors for anti-rabbit IgG and Cy5 fluorescent dye as low as 0.5 ng/µl (∼3.268 nM). We argue that the intrinsic nature of point-to-point and batch-detection methods as showcased in our chip offers advantages over the serial-scanning approach used in traditional scanner systems. In addition, our system is low cost and lightweight.