Optical Biosensors
Porous Silicon (PSi)
Formation and Fabrication
The electrochemical reaction that takes place at the platinum cathode is mainly the reduction of protons to hydrogen gas. For the formation of multilayer PSi structures, improved interface uniformity has been achieved by including a short period of zero bias between individual layers, allowing the hydrogen to diffuse out of the pores and replenish the fluoride at the pore tips [76] . .
Refractive Index of PSi
One of the most commonly used effective medium models is the Bruggeman approach [79], which is given by. A porosity range of 30 to 90% gives an estimated refractive index range of 3.30 to 1.15, depending on the wavelength of light and the morphology of the Psi.
PSi in Label-Free Optical Biosensing
PSi biosensors relying on reflectance-based transduction methods employ photonic structures whose reflectance spectra change upon molecular binding in the porous matrix. In the case of PSi, molecules infiltrate the porous matrix and directly interact with a guided mode.
PSi Photonic Structures
Single Layer Interferometer
A possible method to enhance the optical signal generated by the specific attachment of a small analyte is to label the analyte with quantum dots (QDs) [105]. have shown that QDs can serve both as high refractive index signal amplifiers for reflectance measurements and as fluorescent labels for verifying the incorporation of molecules into the functionalized pores [105]. A more than fivefold improvement in the measured reflection edge shift and an improvement of almost three orders of magnitude in the detection limit for just 6% surface coverage of QDs in the porous matrix were demonstrated for detecting target QD-biotin conjugates with streptavidin probes immobilized in PSi single-layer films. a) Top view and (b) cross-sectional SEM images of a PSi single-layer film. bright areas indicate silicon and the dark areas indicate air.
Waveguide
The resonance angle shifts in response to the change of the effective refractive index of the PSi waveguide layer. The Otto configuration is used for double layer PSi waveguides with an air gap between the waveguide.
Microcavity
Previous work has demonstrated the use of PSi waveguides for the detection of DNA and proteins, establishing the lowest expected detection limit in the nanomolar range on the pore walls, increasing the effective refractive index of PSi, and red-shifting the microcavity resonance to longer wavelengths . Both microcavities and waveguides have the potential to achieve the lowest detection limits [113], with microcavities being easier to implement because the reflection spectrum of a microcavity is measured by a spectrometer with near-normal incident light, while each waveguide requires complex instrumentation for angle-dependent coupling. a) Cross-sectional SEM image of a PSi microcavity. low porosity films and the darker layers indicate high porosity films.
Current Challenges in PSi Biosensing
Surface Passivation
Peroxide-based chemical oxidation is challenged by simultaneous hydrogen bubbling that can cause non-uniformity of the oxide [119]. The corrosion process was further mitigated by reducing the overall concentration of negative charges within the PSi sensing volume, resulting in improved reusability and reliability of the PSi DNA sensor.
Mass Transport Efficiency
To improve mass transport efficiency in PSi, an open-ended PSi membrane has been demonstrated to allow analytes to flow through the pores in microfluidics-based assays and interact more favorably with the inner pore surfaces [149,150]. The flow-through design significantly improves the efficiency of transport of molecules in microcavity structures and reduces the sensor response time compared to the flow-through methods [149,150].
Overview of the Dissertation
The increased concentration of negative charges increases the corrosion rate of the PSi surface. Reflectance spectra of the PSi microcavity measured after electrochemical etching. black line), oxidation (red line), removal of silicon substrate (green line), and flow cell attachment (blue line).
DNA INDUCED CORROSION IN POROUS SILICON BIOSENSORS
Introduction
Previous reports have focused on the rapid oxidation and hydrolysis (i.e., corrosion) of PSi fabricated from p-doped silicon that occurs when PSi films are exposed to solutions containing negative charges. It was suggested that the negative charges on the phosphate backbone of DNA attract positively charged carriers in p-type silicon to the PSi surface, facilitating nucleophilic attack by water molecules that ultimately leads to the dissolution of PSi.
Fabrication of PSi Waveguides
The resulting PSi waveguide layer has a refractive index of approximately 1.77, pore diameters ranging from 15 - 25 nm, and a thickness of approximately 220 nm. The bottom cladding layer has slightly larger pore diameters, a refractive index of approximately 1.32 and a thickness of approximately 1400 nm.
Surface Functionalization
- In Situ DNA Synthesis
- In Situ PNA Synthesis
- Nucleic Acid Sensing
Immediately before hybridization, 10 μL of the 100 μM DNA solution was diluted to a working concentration of 10 μM in 90 μL HEPES buffer. Magnesium chloride hexahydrate (MgCl2 ∙ 6H2O) was dissolved in the HEPES buffer solution used for hybridization of the target DNA with synthesized PNA probes at a concentration of 2 M in order to shield the negative charges on the DNA and evaluate the effect resulting in PSi corrosion.
Prism Coupling Measurements
The resonance angle corresponds directly to the effective refractive index of the waveguide according to the relation. Molecule infiltration inside the pores of the PSi waveguide causes the refractive index of the PSi to increase, leading to a redshift (ie shift to higher angle) of the resonance angle.
Characterization of PSi Passivation
The magnitude of the resonant angle shift correlates and allows quantification of the amount of material added to or removed from the PSi waveguide. Hydrolysis is indicated by the shift of the resonance to lower angles after soaking in DI water.
Influence of Probe DNA on PSi Corrosion
Incubation of the deshielded probe DNA molecules with negatively charged backbones in DI water results in a blue shift to lower resonant angles (dashed lines). The stability of the PSi waveguides functionalized with in situ synthesized probe DNA molecules against corrosion was evaluated by soaking in DI water.
Influence of DNA Hybridization on PSi Corrosion
Thus, without deprotection, the charge-neutral DNA probe molecules do not facilitate the oxidative corrosion of the PSi surface. When the target DNA concentration reaches this threshold, the majority of accessible probe DNA molecules have hybridized to complementary DNA targets within a 1-h measurement.
Mitigating DNA Induced Corrosion in PSi
- DNA Hybridization with Synthesized PNA Probes
- Compensating DNA Charge with Magnesium
The size of the red shift observed after DNA hybridization in the presence of Mg2+ ions is about 1.4 times smaller than the observed results with PNA hybridization, which corresponds to. The detection sensitivity of the well passivated PSi waveguide functionalized with in situ synthesized probe PNA was estimated by exposure to various concentrations of DNA target molecules in the presence of 2 M MgCl2.
Mechanism of Corrosion on Passivated PSi
However, the disappearance of the Si-Hx groups after thermal oxidation indicates good passivation of the PSi waveguide surface. Further oxidation results in condensation of Si-OH bonds on the adjacent surface, forming Si-O-Si bridges, which contribute to the passivation of the PSi surface [177].
Summary
For these conditions, the thickness of the depletion zone ( ) was estimated from. 3.2) The Peclet number ( , defined as the ratio of the time for molecules to reach the sensing surface by diffusion over convection, was calculated from. This annealing step was used to promote the stable formation of the 3-APTES monolayer. The times relatively slow ramps were necessary to ensure that the integrity of the PSi membranes was not compromised due to thermal shock.
COMPUTATIONAL ANALYSIS OF CLOSED-ENDED AND OPEN-ENDED
Introduction
However, the use of open porous membranes in biosensor applications is not common and has not been widely explored. The analysis shows that open pores allow analyte to flow through the pores and greatly reduce the response time and analyte consumption for the detection of large molecules with slow diffusivities compared to closed pores where analytes mostly flow across the pores.
Analytical Calculation of Analyte Transport Efficiency
In the flow-over model, the Peclet number in the channel is calculated to be 1700. The Peclet number within an individual pore (for the flow-through scheme was calculated as
Numerical Model and Simulation
Therefore, laminar flow is used in the simulation to obtain the steady state velocity distribution. First, the steady-state velocity distribution in the flow cell was obtained for laminar flow.
Simulated Transport and Reaction Kinetics
- Velocity Distribution and Analyte Concentration Distribution
- Influence of Flow Velocity
- Influence of Analyte Concentration
- Influence of Analyte Diffusivity and Adsorption Rate
However, in the flow-through scheme with open-ended pores, the required volume of analyte changes little with flow rate. The equilibrium times for porous sensors with closed pores in the overflow scheme and open-ended pores in the flow scheme were investigated for analytes with different diffusivities and adsorption kinetics.
Summary
The resonance wavelength of the PSi microcavity was aligned with the peak emission of the QDs to achieve enhanced photoluminescence (PL) from the QDs immobilized in the microcavity structure [211]. The inset shows camera images of the samples under UV excitation at 365 nm: 1) QDs in an open PSi membrane in the flow-through scheme and 2).
FLOW-THROUGH POROUS SILICON MEMBRANES FOR LABEL-FREE
Introduction
In this chapter, the transport and binding kinetics in these PSi membranes were compared by experiments with those of closed PSi films of comparable thickness in a conventional overflow observation scheme. Quantum dot (QD) based fluorescence measurements were performed to verify the enhanced analyte transport efficiency within open-ended PSi membranes.
Device Fabrication and Measurement
- Electrochemical Etching of PSi Microcavities
- Photolithography Process for PSi Membrane Fabrication
- Microfluidics Integration
- Optical Reflectivity Measurement
The nitride film remaining on the PSi ensures that analytes only flow into the membrane regions of the PSi films. The same contact lithography and RIE was then used to pattern the silicon nitride film and open the windows on the top side of the samples.
Effect of Analyte Size on Sensor Response
Comparison of real-time PSi microcavity response for closed-end pores in the flow scheme and open-end pores in the flow scheme. The time for the PSi microcavity to reach a wavelength shift of 1 nm, an easily measurable value, was a quarter of that when the flow scheme was used.
Surface Sensing
De Stefano, L., Rotiroti, L., Rendina, I., Moretti, L., Scognamiglio, V., Rossi, M., D'Auria, S.: Microsensore ottico a base di silicio poroso per la rilevazione di L- glutammine.
CONCLUSIONS
Summary
In a conventional flow scheme, the pores are closed so that solutes enter and leave through the same end of the pore. In the flow scheme, the pores are open, allowing solutions to enter at one end of the pore and exit through the other end.
Future Research Opportunities
- PSi Microarrays for Multiplexed Biosensing
- Quantum Dot Coupled PSi for Colorimetric Sensing
