The Incorporation of Amplified Metal-Enhanced Fluorescence in a CMOS-Based Biosensor Increased the Detection Sensitivity of a DNA Marker of the Pathogenic Fungus Colletotrichum gloeosporioides

Abstract

Half of the global agricultural fresh produce is lost, mainly because of rots that are caused by various pathogenic fungi. In this study, a complementary metal-oxide-semiconductor (CMOS)-based biosensor was developed, which integrates specific DNA strands that allow the detection of enoyl-CoA-hydratase/isomerase, which is a quiescent marker of Colletotrichum gloeosporioides fungi. The developed biosensor mechanism is based on the metal-enhanced fluorescence (MEF) phenomenon, which is amplified by depositing silver onto a glass surface. A surface DNA strand is then immobilized on the surface, and in the presence of the target mRNA within the sample, the reporter DNA strand that is linked to horseradish peroxidase (HRP) enzyme will also bind to it. The light signal that is later produced from the HRP enzyme and its substrate is enhanced and detected by the coupled CMOS sensor. Several parameters that affect the silver-deposition procedure were examined, including silver solution temperature and volume, heating mode, and the tank material. Moreover, the effect of blocking treatment (skim milk or bovine serum albumin (BSA)) on the silver-layer stability and nonspecific DNA absorption was tested. Most importantly, the effect of the deposition reaction duration on the silver-layer formation and the MEF amplification was also investigated. In the study findings a preferred silver-deposition reaction duration was identified as 5-8 min, which increased the deposition of silver on the glass surface up to 13-times, and also resulted in the amplification of the MEF phenomenon with a maximum light signal of 50 relative light units (RLU). It was found that MEF can be amplified by a customized silver-deposition procedure that results in increased detection sensitivity. The implementation of the improved conditions increased the biosensor sensitivity to 3.3 nM (4500 RLU) with a higher detected light signal as compared to the initial protocol (400 RLU). Moreover, the light signal was amplified 18.75-, 11.11-, 5.5-, 11.25-, and 3.75-times in the improved protocol for all the tested concentrations of the target DNA strand of 1000, 100, 10, 3.3, and 2 nM, respectively. The developed biosensor system may allow the detection of the pathogenic fungus in postharvest produce and determine its pathogenicity state.

Keywords: CMOS biosensor; Colletotrichum gloeosporioides; metal-enhanced fluorescence; pathogenic fungus; postharvest system; silver deposition.

Figure 1

Schematic presentation of the CMOS-based biosensor. (1) Different sections of the complementary metal-oxide-semiconductor (CMOS)-based biosensor platform: (1a) heating element for the silver-deposition reaction; (1b) silver reaction solution that is used to polymerize the silver layer above the glass surface; (1c) reaction tank where the polymerization reaction occurs; (1d) glass tube where the deposition and measurement processes occur; (1e) silver surface nanolayer with immobilized surface DNA strands; (1f) horseradish peroxidase (HRP) enzymatic reaction produces a measurable light signal, amplified with metal-enhanced phenomenon (MEF); (1g) CMOS photodetector that is coupled near the light signal from positive samples and transforms it into measurable electrical current. (2) The DNA immobilization process onto the glass surface: (2a) unmodified glass surface; (2b) activation with piranha solution; (2c) silanization; (2d) nucleation points for further silver deposition; (2e) immobilization of surface DNA strands; (2f) blocking agent (skim milk or bovine serum albumin (BSA)). (3) The measurement process where the surface DNA strand is immobilized onto the silver-deposited glass: (3a) exposure to the target strand; (3b) exposure to the reporter strand that is linked to HRP; (3c) the strands specifically anneal into one complex; (3d) addition of substrate ((1:1) luminol: H₂O₂ (v/v)) produces a measurable light signal.

Figure 2

Picture of the CMOS-based biosensor system setup. (A) the enzymatic reaction of the horseradish peroxidase (HRP) enzyme was activated by the deposition of a 20 µL substrate solution ((1:1) luminol: H₂O₂ (v/v)), (B) while the glass tubes were still placed above the CMOS sensor. (C) A home-made holder from Styrofoam was integrated with the CMOS-based biosensor system setup, which prevents the movement of the glass tube during the measurement process. The light signal was directly monitored and detected by the CMOS sensor.

Figure 3

Effect of different deposition conditions on the efficiency of the silver-layer formation. Various parameters were examined including silver solution temperature and volume, heating mode, and the tank material. The silver-deposition reaction duration was 9 min in all the tested parameters.

Figure 4

Effect of blocking treatment on the silver-layer stability. The influence of two blocking agents ((A) skim milk and (B) bovine serum albumin (BSA)) on the silver-layer stability was examined.

Figure 5

Effect of the blocking step on the DNA nonspecific absorption on the glass surface. The blocking treatment was conducted on the silver-modified glass surfaces, with either bovine serum albumin (BSA) or skim milk. Then, the light signal was compared with the untreated (without blocking agent) glass tubes. After the blocking step, all the tubes were incubated for 1 h with 20 µL 1 µM of the reporter DNA strand linked to horseradish peroxidase (HRP). The light signal was measured after washing, by adding a mixture of 1:1 (v/v) H₂O₂ + luminol solution (HRP substrate). (A) Glass tubes without immobilized surface DNA. (B) The effect of the blocking step on the annealing processes was examined by conducting the blocking treatment on glass tubes with immobilized 20 µL 1 µM surface DNA strand. Then, they were exposed to the reporter strand (DNA-HRP) with or without 20 µL 1 µM of the target DNA strand. After the annealing process, the glass tubes were washed, and the light signal was measured by adding the H₂O₂ + luminol solution (HRP substrate).

Figure 6

Effect of the deposition reaction duration on the silver-layer formation and the metal-enhanced fluorescence (MEF) amplification. Monitoring the influence of the silver-deposition reaction duration on both (A) optical density at 420 nm (OD_{420 nm}), and (B) relative light units (RLU). (C) With a distance lower than 10 nm between the silver-deposited glass surface and the light photons that were generated from the horseradish peroxidase (HRP) enzyme reaction with its substrate, a plasmons transfer occurred that increased the light signal, which was later detected by the complementary metal-oxide-semiconductor (CMOS) sensor. (D) After excessive silver deposition the silver dots formed a silver layer, and the plasmons scattered and reduced the light signal.

Figure 7

Scanning electron microscopy (SEM) characterization of the effect of the deposition time on the formation of silver nano-islands. The surface morphology was characterized by an SEM model MIRA3 from TESCAN (Brno-Kohoutovice, Czech Republic), at a 5 kV accelerating voltage. Before imaging, a thin layer of palladium gold was deposited onto the samples in order to render them electrically conductive and to avoid potential surface charging by the electron beam. The influence of the silver-deposition reaction duration of (A) 1 min; (B) 3 min; (C) 4 min; (D) 6 min; (E) 7 min; (F) 8 min; (G) 9 min; and (H) 10 min, was investigated. A clear correlation is visible between the deposition time and the size and amount of the silver nano-islands. Longer deposition time resulted in significantly-larger silver nano-islands (8 > 7 > 5 > 4 min).

Figure 8

The sensitivity of the CMOS-based biosensor system to Colletotrichum gloeosporioides fungi. The response of the biosensor system to different concentrations of the quiescent marker of Colletotrichum gloeosporioides, a specific sequence of enoyl-CoA-hydratase/isomerase (Cgl_00014454), before and after integrating the conclusions from the optimization steps of the biosensor procedures. (A + B) initial protocol and (C) improved protocol. Negative control (N.C.) was used. For both protocols, the modified glass surfaces were immobilized with 20 µL 1 µM surface DNA strand, exposed to the target DNA strand in different concentrations, and later exposed to 20 µL 1 µM quiescent-stage reporter DNA strand.