Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array

Abstract

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 ~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.

Fig. 1

Strategy for ultrasensitive amperometric detection by the microfluidic immunoarray: (A) a single sensor in the array with capture antibodies. Protein analytes are captured off-line (inside box) on Ab₂-MB-HRP bioconjugates with ~400,000 HRP labels and 120,000 Ab₂. On right in (A), a single sensor is depicted after subsequent Ab₂-MB-HRP magnetically separation and injection into (B) the 8-sensor immunoarray, which is connected to a pump and injector valve (not shown). Amperometric signals are developed at −0.3 V vs. Ag/AgCl by injecting a mixture of H₂O₂ and hydroquinone, and measuring the 8 currents.

Fig. 2

Proteins detected in serum by the microfluidic array using amperometry after incubating of Ab_2-MB-HRP-analytes in measurement chamber for 20 min, then injecting mixture of H₂O₂ and HQ: (A) duplicate responses in simultaneous measurements of a standard mixture of 10 fg mL⁻¹ IL-6, 15 fg mL⁻¹ IL-8, 25 fg mL⁻¹ VEGF, and 60 fg mL⁻¹ VEGF-C illustrating reproducibility, (B) responses to VEGF in mixtures of biomarker proteins (peaks for VEGF were extracted from four-protein determinations and presented together), (C–F) Immunoarray calibration plots of standard mixtures in calf serum for IL-6 (C), IL-8 (D), VEGF (E), VEGF-C (F), using background corrected peak currents. Standard deviations correspond to 2 sensors each on three separate arrays (n=6).

Fig. 3

Immunoarray and ELISA results for (A–D) conditioned media for cells (Cal27, HaCaT, HN12, HN13, HN17, HN30, NOKsi, HNOK26, NOK6TK4, and NOK16TK4) (A) IL-6, (B) IL-8, (C) VEGF, (D) VEGF-C. * levels of individual biomarker were below detection limit of ELISA, and ** level of protein above range for detection by commercial ELISA. (E) NOKsi cells under normoxia (−) and hypoxia (+) conditions for IL-6, IL-8, VEGF, and VEGF-C detection. Error bars represent standard deviation for immunoarray (n=6) and average deviation for ELISA (RSD ± 10%).

Fig. 4

Receiver operating characteristic (ROC) curves for serum assays for (A) IL-6, AUC 0.86 ( ), IL-8 with AUC 0.83 ( ), VEGF with AUC 0.95 ( ), VEGF-C with AUC 0.83 ( ), and (B) mean normalized value for all 4 proteins, with AUC 0.96.

Fig. 5

Statistical analysis of array data from various cancer classes: (A) vertical scatter plots of normalized biomarker level for cancer-free control (con), early (Tis, T1, T2) and late (T3, T4) stage tumor stages showing means and standard deviations (error bars), (B) ROC curves comparing cancer-free vs. early stage ( ); early vs. late stage cancer ( ); and cancer-free vs. late stage ( ).