44 research outputs found
Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array
Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg.mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400 000 enzyme labels and similar to 100 000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89 and specificity of 98 for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid Serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers. This record was migrated from the OpenDepot repository service in June, 2017 before shutting down
Ultrasensitive nanostructured immunosensor for stem and carcinoma cell pluripotency gatekeeper protein NANOG
AIMS: To develop an immunosensor for ultrasensitive detection of the NANOG protein. NANOG regulates pluripotency in stem cells and some cancer cells. This article reports the first electrochemical immunosensor for ultrasensitive detection and absolute quantification of the NANOG protein. The sensor features dense capture antibody-coated gold nanoparticle layers on a pyrolytic graphite underlayer. MATERIALS & METHODS: Two separate multilabel detection strategies were used to achieve moderate and ultra-high sensitivity. RESULTS: Good sensitivity was achieved for NANOG over the concentration range 0.1–160 pg/ml. The moderate sensitivity approach gave a detection limit of 25 pg/ml, while the ultrasensitive method achieved a 250-fold lower detection limit of 0.1 pg/ml. Amounts of NANOG detected in human embryonic stem cell lysates correlated well with qualitative western blots and mRNA expression. CONCLUSION: The electrochemical gold nanoparticle immunosensor is suitable for measuring NANOG protein expression in stem and carcinoma cell tissue lysates at very low levels
Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array
Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg.mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400 000 enzyme labels and similar to 100 000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid Serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers
Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array
Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg.mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400 000 enzyme labels and similar to 100 000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid Serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers
Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array
Multiplexed biomarker protein detection holds unrealized
promise
for clinical cancer diagnostics due to lack of suitable measurement
devices and lack of rigorously validated protein panels. Here we report
an ultrasensitive electrochemical microfluidic array optimized to
measure a four-protein panel of biomarker proteins, and we validate
the protein panel for accurate oral cancer diagnostics. Unprecedented
ultralow detection into the 5–50 fg·mL<sup>–1</sup> range was achieved for simultaneous measurement of proteins interleukin
6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C
in diluted serum. The immunoarray achieves high sensitivity in 50
min assays by using off-line protein capture by magnetic beads carrying
400 000 enzyme labels and ∼100 000 antibodies. After
capture of the proteins and washing to inhibit nonspecific binding,
the beads are magnetically separated and injected into the array for
selective capture by antibodies on eight nanostructured sensors. Good
correlations with enzyme-linked immunosorbent assays (ELISA) for protein
determinations in conditioned cancer cell media confirmed the accuracy
of this approach. Normalized means of the four protein levels in 78
oral cancer patient serum samples and 49 controls gave clinical sensitivity
of 89% and specificity of 98% for oral cancer detection, demonstrating
high diagnostic utility. The low-cost, easily fabricated immunoarray
provides a rapid serum test for diagnosis and personalized therapy
of oral cancer. The device is readily adaptable to clinical diagnostics
of other cancers
Automated Multiplexed ECL Immunoarrays for Cancer Biomarker Proteins
Point-of-care
diagnostics based on multiplexed protein measurements
face challenges of simple, automated, low-cost, and high-throughput
operation with high sensitivity. Herein, we describe an automated,
microprocessor-controlled microfluidic immunoarray for simultaneous
multiplexed detection of small protein panels in complex samples.
A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell
detection array to achieve sensitive detection of four prostate cancer
biomarker proteins in serum. The proteins are prostate specific antigen
(PSA), prostate specific membrane antigen (PSMA), platelet factor-4
(PF-4), and interlukin-6 (IL-6). The six channel system is driven
by integrated micropumps controlled by an inexpensive programmable
microprocessor. The reagent delivery cassette and detection array
feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall
carbon nanotube forests were grown in printed microwells on a pyrolytic
graphite detection chip and decorated with capture antibodies. The
detection chip is housed in a machined microfluidic chamber with a
steel metal shim counter electrode and Ag/AgCl reference electrode
for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent
cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica–antibody
detection nanoparticles sequentially. An onboard microcontroller controls
micropumps and reagent flow to the detection chamber according to
a preset program. Detection employs tripropylamine, a sacrificial
reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was
measured by a CCD camera. Ultralow detection limits of 10–100
fg mL<sup>–1</sup> were achieved in simultaneous detection
of the four protein in 36 min assays. Results for the four proteins
in prostate cancer patient serum gave excellent correlation with those
from single-protein ELISA