"Silicon-On-Insulator"-Based Biosensor for the Detection of MicroRNA Markers of Ovarian Cancer

Abstract

Ovarian cancer is a gynecological cancer characterized by a high mortality rate and tumor heterogeneity. Its early detection and primary prophylaxis are difficult to perform. Detecting biomarkers for ovarian cancer plays a pivotal role in therapy effectiveness and affects patients' survival. This study demonstrates the detection of microRNAs (miRNAs), which were reported to be associated with ovarian cancer tumorigenesis, with a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). The advantages of the method proposed for miRNA detection using the SOI-NW biosensor are as follows: (1) no need for additional labeling or amplification reaction during sample preparation, and (2) real-time detection of target biomolecules. The detecting component of the biosensor is a chip with an array of 3 µm wide, 10 µm long silicon nanowires on its surface. The SOI-NW chip was fabricated using the "top-down" method, which is compatible with large-scale CMOS technology. Oligonucleotide probes (oDNA probes) carrying sequences complementary to the target miRNAs were covalently immobilized on the nanowire surface to ensure high-sensitivity biospecific sensing of the target biomolecules. The study involved two experimental series. Detection of model DNA oligonucleotides being synthetic analogs of the target miRNAs was carried out to assess the method's sensitivity. The lowest concentration of the target oligonucleotides detectable in buffer solution was 1.1 × 10^-16 M. In the second experimental series, detection of miRNAs (miRNA-21, miRNA-141, and miRNA-200a) isolated from blood plasma samples collected from patients having a verified diagnosis of ovarian cancer was performed. The results of our present study represent a step towards the development of novel highly sensitive diagnostic systems for the early revelation of ovarian cancer in women.

Keywords: biomarkers; biosensor; miRNA; nanowire; oDNA; ovarian cancer; silicon-on-insulator.

Figure 1

The structure of molecular probes probe_1 (a), probe_2 (b), and probe_3 (c) obtained using the RNAfold source (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi; accessed on 8 December 2022).

Figure 2

(a) A schematic view of the analytical module of a SOI-NW biosensor. Numbers indicate the key components of the module: (1) a stirrer; (2) a platinum electrode; (3) a measuring cell; (4) a SOI-NW chip; (5) a chip holder; (6) a peristaltic pump; (7) a waste container; (8) a ten-channel data collection and storage system, and (9) a PC. (b) A photographic image of the experimental setup.

Figure 3

(a) The model of the SOI-NW chip being used. (b) An optical image of the surface of the SOI-NW chip. The arrangement of nanowires on the chip surface is outlined with orange color. (c) A SEM image of an individual silicon nanowire.

Figure 4

Scanning electron microscopy (SEM) image of the surface morphology of a silicon nanowire.

Figure 5

A schematic representation of the principle of the functionalization of the sensor surface. The functionalization comprised chemical modification and sensitization of the SOI-NW chip surface, which were performed in order to provide reliable detection of the target biomolecules.

Figure 6

Typical volt–ampere characteristics recorded for one and the same nanowire on the SOI-NW chip with n-type conductivity. Measurement conditions: 1 mM PPMB; V_g = 0 – 60 V, V_ds = 0.2 V; the oDNA probe probe_1 was covalently immobilized on the nanowire. The VACs were recorded before the nanowire functionalization (blue curve), after the nanowire silanization (pink curve), and after its sensitization with oDNA probes (violet curve).

Figure 7

Typical sensogram curves recorded upon the biosensor-based detection of synthetic oDNA analogues of target miRNAs, oligonucleotides CS 1 (a), CS 2 (b), and or CS 3 (c) in buffer solutions. Experimental conditions: SOI-NW chip with n-type conductivity; nanowires were sensitized with oDNA probes (probe_1, probe_2, and probe_3); 1 mM PPMB, V_g = 50 V, V_ds = 0.2 V; solution volume in the measuring cell was 450 µL. Concentrations of the solutions of oDNA targets in the cell were as follows: 1.1 × 10^–17 M (1, pink curve); 1.1 × 10^–16 M (2, violet curve); 1.1 × 10^–15 M (3, olive curve); and 1.1 × 10^–14 M (4, blue curve). The number of technical replicates, n = 3. Arrows indicate the time point of addition of the solution of the oDNA target into the cell, and the washing with pure buffer.

Figure 8

Typical sensorgram curves recorded in the blank experiments, in which the oDNA-free buffer was added into the measuring cell instead of target oDNA solution. Experimental conditions: nanowires with immobilized oDNA probes (probe_1, probe_2, and probe_3); 1 mM PPMB; V_g = 50 V, V_ds = 0,2 V; solution volume in the measuring cell was 450 µL. Number of technical replicates, n = 5. The arrow indicates the time point when the buffer under study (SAMPLE) was added.

Figure 9

The results of biospecific detection of miRNAs isolated from blood plasma samples. Experimental conditions: SOI-NW chip with n-type conductivity; nanowires with immobilized oDNA probes: probe_1 (a), probe_2 (b), and probe_3 (c); 1 mM PPMB; V_g = 50 V, V_ds = 0.2 V; liquid volume in the measuring cell was 107 µL. Analyzed samples: samples No. 8, No. 10, No. 14, and No. 15 isolated from blood plasma of patients with ovarian cancer (olive, purple, orange, and red curves without markers); control sample No. 80 isolated from the plasma sample collected from a patient with breast cancer (blue curve, triangles); control samples No. 1 and No. 2 isolated from plasma samples collected from healthy volunteers (black curve, circles and pink curve, squares, respectively). The arrow indicates the time point when the analyzed solution containing isolated miRNAs was added.

Figure 10

Typical sensogram obtained in the additional control experiment. The signal from the nanowire sensitized with immobilized oDNA probe not specific to any target miRNA (probe_5) was recorded in the case of adding miRNA solution isolated from the plasma sample of a patient with a verified diagnosis of ovarian cancer (No. 20). Experimental conditions: SOI-NW chip with n-type conductivity; the nanowire with immobilized oDNA probe (probe_5); 1 mM PPMB; pH 7.4; V_g = 50 V, V_ds = 0.4 V; the total solution volume in the cell was 107 µL. Number of technical replicates, n = 3. Arrows indicate the time points of addition of the analyzed miRNA solution and the pure washing buffer into the cell.