Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors
<p>Schematic drawing of the four steps of the surface functionalization protocol leading to the target binding (fifth step). “R” refers to the silane residue, i.e., the epoxy group when GPTMS is used, and the thiol group when MPTMS is used.</p> "> Figure 2
<p>Optical measurement setup. (<b>a</b>) Schematic representation of the optical measurement setup (fluidic in blue, optical fibers in red) and magnification showing the fluidic chamber superimposed on a sketch of MRRs and optical waveguides (chip detail). The four central microrings (not hatched in the figure) were used for the binding measurements reported in this work. (<b>b</b>) MRR resonance spectrum showing the optical power (“through” direct output) as a function of the input wavelengths. The different colors refer to the different resonances.</p> "> Figure 3
<p>AFM images of plane silicon surfaces. (<b>a</b>) Silicon without treatment; (<b>b</b>) p1g01 treatment; (<b>c</b>) p1m01 treatment; (<b>d</b>) p1m1 treatment. The false color scale ranges from −0.5 to +3.5 nm, while scale bars represent 200 nm in (<b>a</b>,<b>b</b>), and 100 nm in (<b>c</b>,<b>d</b>).</p> "> Figure 4
<p>Density of aptamer molecules on silicon nitride plane surfaces at different concentrations of aptamers. (<b>a</b>) Amino-terminated or thiol-terminated and fluorescent a-spike aptamers bound at different concentrations to p1g01 (black squares) and p1m1 (red circles) surfaces, respectively. (<b>b</b>) Comparison between thiol-terminated and fluorescent a-spike and a-CRP aptamers bound to p1m1 at a 1 µM concentration. Means and standard deviations of at least three independent surfaces are reported.</p> "> Figure 5
<p>Setup of the aptamer binding on MPTMS silanized surfaces: time of reaction. Aptamer density on silicon nitride surfaces, which were functionalized as p1m1 and treated with 1 µM of a-spike or a-CRP aptamer at room temperature for different times.</p> "> Figure 6
<p>Surface density of the a-spike aptamer on p1m1 silicon nitride surfaces, passivated with 1 mM MCH for different lengths of time. Time 0 refers to non-passivated surfaces.</p> "> Figure 7
<p>AFM analysis of photonic chip waveguides. (<b>a</b>) Typical image of a chip waveguide. Scale bar: 1000 nm. False color scale range from −45 to +345 nm. The yellow line indicates the section through which the height profile shown in panel (<b>b</b>) is drawn. (<b>c</b>) Representative image of the roughness of the top of the guide. Scale bar: 30 nm. False color scale from −3 to +4 nm.</p> "> Figure 8
<p>Confocal representative image of the fluorescent a-CRP aptamer immobilized on an optical microdevice. The area of the sensing window is shown, comprising waveguides that are more fluorescent than the surrounding area due to the difference in materials. Images are acquired with the 63× objective in liquid. The white bar refers to 50 µm.</p> "> Figure 9
<p>Optical detection of thrombin. Kinetics of thrombin binding on p1m1 functionalized photonic chip surfaces, with the a-Thr (black curve) or the NS (red curve) aptamer. Chip surfaces were passivated with 1 mM MCH. Both buffer and 100 nM thrombin solutions were added, with 0.05% Tween 20. Data are means of the four microrings, while standard deviations are represented as dashed curves.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Setup of the Functionalization Protocol
2.3. Characterization of the Functionalization Steps
2.4. Optical Setup and Measurements
3. Results and Discussion
3.1. Activation of Silicon Surfaces by Plasma Treatments
3.2. Development of the Silanization Protocol
3.3. Binding of Aptamers on the Silanized Surfaces
3.4. Passivation of the Functional Surfaces
3.5. Functionalization Protocol Applied to Photonic Chips
3.6. Measures of Protein Targets with the Optical Setup
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Plasma Treatment Name | Gas | Power (W) | Time (min) |
---|---|---|---|
p1 | argon | 10.5 | 1 |
p2 | oxygen | 50 | 2 |
Surface Name | Plasma Treatment | Silanization Name | Silane | % v/v | Sonication |
---|---|---|---|---|---|
p1g1 | p1 | g1 | GPTMS | 1 | - |
p1g1s | p1 | g1s | GPTMS | 1 | 10’ |
p1g01 | p1 | g01 | GPTMS | 0.1 | - |
p1g01s | p1 | g01s | GPTMS | 0.1 | 10’ |
p2g1 | p2 | g1 | GPTMS | 1 | - |
p2g1s | p2 | g1s | GPTMS | 1 | 10’ |
p2g01s | p2 | g01s | GPTMS | 0.1 | 10’ |
p1m1 | p1 | m1 | MPTMS | 1 | - |
p1m1s | p1 | m1s | MPTMS | 1 | 10’ |
p1m01 | p1 | m01 | MPTMS | 0.1 | - |
p1m01s | p1 | m01s | MPTMS | 0.1 | 10’ |
p2m1 | p2 | m1 | MPTMS | 1 | - |
p2m1s | p2 | m1s | MPTMS | 1 | 10’ |
p2m01 | p2 | m01 | MPTMS | 0.1 | - |
p2m01s | p2 | m01s | MPTMS | 0.1 | 10’ |
Name | Sequence (5′–3′) | Target | 5′ Modification | 3′ Modification |
---|---|---|---|---|
a-CRP | CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G | CRP | 5ThioMC6-D/ | /36-TAMSp/ |
a-spike | CAG CAC CGA CCT TGT GCT TTG GGA GTG CTG GTC CAA GGG CGT TAA TGG ACA | Spike protein | /5AmMC12/; /5ThioMC6-D/ | /36-TAMSp/ |
a-Thr | AG TCC GTG GTA GGG CAG GTT GGG GTG ACT | Thrombin | /5ThioMC6-D/ | |
NS | ATC TAC GAA TTC ATC AGG | - | /5ThioMC6-D/ |
Elemental Composition | ||||
---|---|---|---|---|
Surfaces | O % | C % | Si % | Roughness (nm) |
p1 | 31.1 | - | 68.9 | 0.24 ± 0.04 |
p2 | 41.1 | 5.3 | 53.6 |
Surface | Elemental Composition | Roughness | Contact Angle | Thiol Density | |||
---|---|---|---|---|---|---|---|
(GPTMS) | O % | C % | Si % | S (%) | (nm) | (°) | (n × 1014/cm2) |
p1g1 | 3.6 ± 2.8 | 57.3 ± 1.1 | - | ||||
p1g1s | 30.6 | 22.4 | 47.0 | - | 0.7 ± 0.1 | 58.0 ± 1.2 | - |
p1g01 | 31.7 | 18.0 | 50.3 | - | 0.7 ± 0.1 | 62.6 ± 2.0 | - |
p1g01s | 30.9 | 19.5 | 49.6 | - | 0.35 ± 0.03 | 52.5 ± 0.7 | - |
p2g1 | 47.5 | 15.3 | 37.2 | 1.1 ± 0.7 | 55.7 ± 1.3 | - | |
p2g1s | 37.0 | 20.1 | 42.9 | - | 0.50 ± 0.04 | 52.2 ± 0.7 | - |
p2g01s | 36.3 | 21.5 | 42.2 | - | 0.43 ± 0.03 | 52.0 ± 1.1 | - |
(MPTMS) | |||||||
p1m1 | 29.3 | 15.7 | 52.4 | 2.6 | 0.70 ± 0.06 | 60.1 ± 2.3 | 4.0 ± 0.9 |
p1m1s | 29.8 | 15.9 | 52.8 | 1.5 | 0.9 ± 0.1 | 46.0 ± 2.7 | 3.6 ± 0.2 |
p1m01 | 1.1 | ||||||
p1m01s | 28.7 | 13.2 | 56.1 | 2.0 | 0.61 ± 0.06 | 44.6 ± 2.3 | 0.5 |
p2m1 | 60.2 ± 2.1 | 4.2 ± 0.6 | |||||
p2m1s | 35.6 | 15.5 | 45.6 | 3.3 | 0.82 ± 0.07 | 45.8 ± 2.5 | 2.5 ± 0.5 |
p2m01 | 1.0 | ||||||
p2m01s | 36.0 | 14.0 | 48.5 | 1.5 | 0.7 ± 0.1 | 45.3 ± 1.0 | 0.2 |
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Ardoino, N.; Lunelli, L.; Pucker, G.; Vanzetti, L.; Favaretto, R.; Pasquardini, L.; Pederzolli, C.; Guardiani, C.; Potrich, C. Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors. Sensors 2024, 24, 3107. https://doi.org/10.3390/s24103107
Ardoino N, Lunelli L, Pucker G, Vanzetti L, Favaretto R, Pasquardini L, Pederzolli C, Guardiani C, Potrich C. Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors. Sensors. 2024; 24(10):3107. https://doi.org/10.3390/s24103107
Chicago/Turabian StyleArdoino, Niccolò, Lorenzo Lunelli, Georg Pucker, Lia Vanzetti, Rachele Favaretto, Laura Pasquardini, Cecilia Pederzolli, Carlo Guardiani, and Cristina Potrich. 2024. "Optimization of Surface Functionalizations for Ring Resonator-Based Biosensors" Sensors 24, no. 10: 3107. https://doi.org/10.3390/s24103107