Surface Plasmon Resonance (SPR) biosensor has been widely applied in bio-sensing for diagnostic purposes due to the real-time/label-free detection capacity as well as for its high sensitivity28, 29. The most common and widely used SPR optical set-up is the
“Kretschmann configuration” as shown in Fig. 17. In such optical configuration, an
optical beam source illuminates a metal layer through a prism coupler in order to reach the plasmonic resonance angle. At this resonance angle, a large portion of the beam is absorbed; therefore, the intensity of the reflected beam is attenuated to a large extent.
When a target bio-marker is captured by the probe molecule on the surface of the plasmonic layer, the refractive index in the vicinity of the surface is affected resulting in a shift of the resonance angle. The photo-detector, which is fixed at the resonance angle, then records an increase in optical intensity. In this way, the bio-chemical reaction information is transformed into optical intensity data.
Kretschmann configuration SPR, or so-called amplitude based SPR, has numerous advantages. First, it can study real-time biochemical kinetics without the need of molecular tagging (e.g, fluorescence). Secondly, it is mechanically robust and simple.
Only few relevant optical components are needed to build the system. However, Kretschmann configuration SPR has also its shortcomings. It is known that amplitude based SPR has relatively lower sensitivity as compared to a phase-sensitive SPR counter
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parts24, 30. Based on empirical evidences and theoretical derivation, many reviews suggest that the minimal refractive index resolution (∆𝑛𝑚𝑖𝑛) of amplitude based SPR is around 10-5 Refractive Index Unit (RIU)31. Reports also suggest that the SPR limitation comes from intrinsic noise level.
As a result, amplitude based SPR is expected to experience sensitivity issue in low concentration sample (pM to fM range) or low purity sample. Even with proper blocking agent, non-specific bindings during detection may reduce sensitivity over 100 times.
Therefore, improving SPR sensitivity is a real challenge for on-site application but also for clinical and laboratory use.
Many past literature indicate that phase interrogation SPR can be considered as potential candidate32-34 to address such sensitivity issue. Considering the phase change over refractive index (∆𝜙/Δ𝑛) and the obtained Δ𝑛𝑚𝑖𝑛, the phase interrogation SPR has been claimed to be 100 times more sensitive as compared to its amplitude counterparts.
To be more specific, the phase interrogation SPR has been reported to reach ∆𝑛𝑚𝑖𝑛 of 10-8 RIU. Nevertheless, phase interrogation SPR also suffer from its own shortcomings.
Fig. 17 SPR optical set-up based on Kretschmann Configuration.
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It requires multiple optoelectronic components such as beam splitters, and usually a frequency or phase modulator using a piezo-actuator, an Acousto-Optical Modulator (AOM), a Photo-elastic Modulator (PEM) or a wavelength tunable optical source. These instruments increase complexity, weight and price of the device. Besides drawbacks mentioned above, phase interrogation makes the instrument highly vulnerable to mechanical vibrations. These setbacks have not just limited the on-site application of phase sensitive SPR. In the SPR market, which has BiacoreTM, Bio-suplarTM, SensiaTM, GWCTM as major players, there are nearly no commercially available phase sensitive SPR.
In other words, the above-mentioned limitation has entirely prevented the commercialization phase interrogation SPR.
Based on the discussion above, we consider that it is desirable to have a new optical scheme that offers the simplicity of Kretschmann configuration while providing a phase interrogation measurement. Most importantly, a monolithic design should be used to strongly minimize the effect of mechanical vibration. In this way, we can have a SPR set-up that is mechanically suitable for point-of-care application and has enough sensitivity to address the low pre-processing level of the sample.
To fulfill such purposes, we introduce a new type of SPR optical set-up, which we will refer to as “Shearing Interferometry based Surface Plasmon Resonance Biosensor (SiSPR biosensor)”. The SiSPR is realized by three key components. The first key component is
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the plasmonic chip combining the traditional metal layer and a monolithic interferometer.
The second key factor is the use of a low cost spatially and spectrally single-mode laser diode which has a modest wavelength tunability of about 0.079% of its nominal wavelength. The last key component is a newly devised phase retrieval method.
The SiSPR chip and a brief overview of the system can be seen in Fig. 18. The chip is composed of a glass slide sandwiched by a plasmonic layer and a reflective layer. The extra-reflective layer, lead to division and recombination of the beam wavefront. As a result, a shearing interferometer is integrated within a plasmonic chip.
The monolithic SiSPR chip provides the merit of being less vulnerable to mechanical vibration than other conventional homodyne interferometers. To extract the phase, a sinusoidal modulation of the laser wavelength is used to induce Sinusoidal Phase Modulation (SPM). Although not trivial, several options exist to recover the phase
Fig. 18 An overview of SiSPR optical set-up.
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information in presence of a sinusoidal phase modulation. However, the SPM induced by a current modulation makes the phase extraction especially delicate, since a non-negligible amplitude modulation is also induced by this current modulation, exactly at the same frequency.
To solve this problem, we devised a new phase extraction method for SiSPR, where the amplitude of the current modulation "∆i(t)", the phase modulation depth "∆ϕ𝑎", and
the wavelength-to-current sensitivity factor “S” are the major parameter to consider for extracting the SiSPR phase “ϕ𝑆𝑖𝑆𝑃𝑅". The merits of SiSPR are listed in Table 2 in order
to illustrate the points discussed above. In the next chapter (chapter 7), a literature review is provided. This review focus mainly on the limitations of homodyne interferometry (including few related patents), the ∆𝑛𝑚𝑖𝑛 reported for SPR in past literature and, finally.
Amplitude based SPR Phase sensitive SPR SiSPR 1.Sensitivity
(RIU) Moderate (10-5) High (10-7) High (10-7) 2.Mechanical
noise Not affected Affected Not affected
3.Cost low High Moderate
Table 2 Comparison of SiSPR with conventional amplitude or phase sensitive SPR
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The notion of “sensitivity” and the method of estimating ∆𝑛𝑚𝑖𝑛 remain topics of debate
among different SPR reports. Through selected references, we will detail how these quantities are defined and discuss the possible issues related to the definition. In chapter 8, we detail the SiSPR technology. Starting with the basic working principle of SiSPR, we first provide a wave optics description of the SiSPR. Then, we discuss the angle dependence of the optical path difference in this system and we then describe the fabrication process of the plasmonic chip. At the end of chapter 8, the proposed phase extraction method is derived and analyzed. In order to keep concise the thesis, the main focus in this chapter is to introduce the work flow as well as five related phase extraction parameters. A more detailed derivation is placed in appendix. In chapter 9, the results of SiSPR experiments are finally revealed. Optical images of the SiSPR interferences are provided to validate the above-mentioned working principle. The measurement of “S”
and corresponding “∆ϕ𝑎” are demonstrated. To evaluate the system sensitivity in terms of Δϕ/Δn and ∆𝑛𝑚𝑖𝑛, a sensing experiment on a series of glucose reference solution is
demonstrated. We then compare ΔI/Δn (sensitivity of detection from amplitude information) with Δϕ/Δn (sensitivity of detection from phase
information) and estimate the ultimate sensitivity advantage of SiSPR. In the final part of the thesis, we perform a surface modification with a 40 mer “tro4”
aptameric probe and carried out preliminary protein sensing. The most technical
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part for potable prototype integration is demonstrated in appendix.
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