General principle of operation
In this study, we sought to design a biosensor that combines photonic-to-RF conversion and antigen-antibody interaction in an OFC. The biosensing OFC operates through three steps: (1) antigen-antibody interactions on the antibody-modified sensor surface, (2) RI-dependent optical spectrum shift of OFC provided by the intracavity MMI fibre sensor [31, 32, 37], and (3) photonic-to-RF conversion by the wavelength dispersion of the fibre cavity [33–35], as depicted in Fig. 1a. In step (1), the selective combination of a target antigen with the corresponding antibody changes the effective RI near the sensor surface depending on the antigen concentration. In step (2), since the intracavity MMI fibre sensor transmits only certain wavelength (λMMI) light based on its RI due to MMI and the Goos-Hänchen shift, the OFC shows an RI-dependent and hence an antigen-concentration-dependent shift in the optical spectrum. Simultaneously, the intracavity MMI fibre sensor enhances the sensing sensitivity by multiple interactions between light and sample inside the OFC cavity. The functions of steps (1) and (2) are implemented by an intracavity MMI fibre sensor with antibody surface modification, as shown in Fig. 1b. In step (3), the antigen-concentration-dependent shift in the optical spectrum is converted to a shift in the optical cavity length nL, where n and L are the RI and the physical length of the OFC cavity fibre, via the wavelength dispersion of the fibre cavity. Finally, the change in the antigen concentration can be read out as the frep shift via frep = c/nL, where c is the velocity of light in a vacuum. Importantly, the frep linewidth achieves down to below 1 Hz [33], which is smaller than frep shift expected due to the antigen concentration change.
Temperature Drift In The Single-comb Configuration
We first evaluated the dependence of frep in a single sensing OFC on cavity temperature because the temperature disturbance to the fibre OFC cavity fluctuates frep via thermal expansion or shrinkage of nL. To this end, we measured the temporal fluctuation in the frep of the single-comb configuration of sensing OFC under an uncontrolled cavity temperature, as shown in Fig. 2a, whose details are given in the Materials and Methods section. We set the centre optical wavelength lMMI of 1556.6 nm and the frequency spacing frep of 31.7 MHz for stable mode-locked oscillation with the intracavity MMI fibre sensor. Pure water was used for a standard sample with a stable RI, and placed in a glass sample cell together with the MMI fibre sensor without the surface modification for RI sensing. The output light from the OFC was detected by a photodetector (PD), and frep was measured by an RF frequency counter synchronized to a rubidium frequency standard working in the RF band. Figure 2b shows the frep shift (δfrep, blue line) when the cavity temperature (orange line) changed over a range of 1°C. δfrep represents the frequency deviation from the initial measurement value. The temporal behaviour of δfrep in synchronization with the cavity temperature indicated a temperature sensitivity of approximately − 400 Hz/°C. Thus, the cavity-temperature-dependent frep fluctuation is considerably larger than the sample-concentration-dependent frep shift in biosensing (typically, a few to a few tens Hz). Although the cavity temperature was actively controlled within a range of 25.0 ± 0.1°C in the following experiments (see the Materials and Methods section), it is still insufficient to supress the cavity-temperature-dependent frep fluctuation (= 400 Hz/°C × 0.1°C = 40 Hz) below the sample-concentration-dependent frep shift. Thus, to further reduce the temperature drift, we applied a dual-comb configuration for active-dummy compensation of the temperature drift together with the active control of cavity temperature, as described in the following subsection.
Active-dummy Compensation Of The Temperature Drift With The Dual-comb Configuration
A dual-comb configuration [36] with an active sensing OFC with a frequency spacing of frep1 and a dummy sensing OFC with a frequency spacing of frep2 was adopted to compensate for the temperature drift. Figure 3a shows a schematic drawing of the dual-comb configuration, in which a pair of fibre OFC cavities were arranged in a temperature-controlled box so that they were affected by similar temperature fluctuations. In this configuration, although frep1 and frep2 fluctuate depending on the residual fluctuation of cavity temperature via thermal expansion or shrinkage of nL, their fluctuations are similar because they experience the same thermal disturbances. Therefore, the frequency difference ∆frep between frep1 and frep2 remains constant regardless of the temperature drift of frep1 and frep2. Thus, when the active sensing OFC evaluates a sample solution in a certain temperature environment and the dummy sensing OFC evaluates a reference material in the same temperature environment, ∆frep reflects the sample concentration without influence from temperature drift. In other words, a one-to-one correspondence between ∆frep and the antigen concentration independent of temperature drift is established. Figures 3b, 3c, and 3d show the MMI and sample cell for dual-comb RI sensing of pure water, dual-comb RI sensing of glycerol solution, and dual-comb biosensing of the SARS-CoV-2 N protein antigen, respectively. Table 1 summarizes lMMI, frep1, frep2, ∆frep, the MMI, and the sample cell used in the following three dual-comb sensing experiments; these values were selected for stable operation and better temperature compensation. A pair of output lights from the active and the dummy sensing OFCs is detected by a pair of photodetectors (PDs). Their frequency signals (= frep1 and frep2) and a frequency difference between them (= ∆frep = frep1 - frep2) are measured by the RF frequency counter. Details of the dual-comb biosensing technique are given in the Materials and Methods section, together with details on the experimental and analytical methodology employed for all measurements.
Table 1
Experimental settings of dual-comb RI sensing and biosensing.
Experiments | lMMI (nm) | frep1 (MHz) | frep2 (MHz) | ∆frep (kHz) | Active MMI | Dummy MMI | Sample cell |
Dual-comb RI sensing of pure water | 1556.6 | 31.7 | 32.5 | -851.9 | No surface modification | Single (Fig. 3b) |
Dual-comb RI sensing of glycerol solution | 1556.6 | 31.7 | 32.5 | -851.9 | No surface modification | Dual (Fig. 3c) |
Dual-comb biosensing of SARS-CoV-2 N protein antigen | 1556.6 | 29.6 | 29.7 | -88.6 | Surface modification of antibody | No surface modification of antibody | Single (Fig. 3d) |
The blue and green lines in Fig. 4 show the temporal shifts in frep1 and frep2, namely, δfrep1 and δfrep2, respectively, when pure water was used as a sample for both the active and dummy sensing OFCs without surface modification (see Table 1 and Fig. 3b). δfrep1 and δfrep2 suffered from a temperature drift of over − 38 Hz due to an increase in the cavity temperature; however, they behaved almost the same in terms of drift. The resulting ∆frep shift (δ∆frep) was stable, with a variation up to 1.18 Hz, as shown by the red line in Fig. 4; this variation is equivalent to a temperature stability within 0.0030°C much better than that by the temperature controller of fibre OFC cavity. This level of ∆frep stability is sufficient for precise measurement of the sample-concentration-dependent ∆frep shift.
We next tested active-dummy temperature compensation for RI sensing of a liquid sample different from the reference sample. For RI sensing, the active and dummy sensing OFCs have no surface modification (see Table 1 and Fig. 3c). We used glycerol solutions consisting of glycerine and pure water at different ratios, corresponding to different RIs, as target samples in the active sensing OFC. This sample is easy to prepare and stable as it does not volatilize. We prepared six samples with different RIs (= 0 vol%, 1 vol%, 2 vol%, 3 vol%, 4 vol%, and 5 vol%, corresponding to 1.3334 RIU, 1.3350 RIU, 1.3366 RIU, 1.3382 RIU, 1.3398 RIU, and 1.3414 RIU; see colour-highlighted zones in Figs. 5a and 5b) because their expected frep shift is comparable to the concentration-dependent frep shift caused by the SARS-CoV-2 N protein antigen. We exchanged the sample by using a peristaltic pump (see grey zones in Figs. 5a and 5b). Additionally, pure water (a 0 vol% glycerol solution, corresponding to 1.3334 RIU) was used as a reference sample in the dummy sensing OFC. To prevent the temperature of the pure water in the dummy sample cell from increasing during repeated measurements, the pure water sample was exchanged with a new pure water sample with another peristaltic pump when the target sample was exchanged with a new RI glycerol sample. The blue and green lines in Fig. 5a represent δfrep1 and δfrep2 as the concentration of the glycerol solution increased from 0 vol% to 5 vol%. δfrep2, in the dummy sensing OFC, exhibited a slow drift with some rapid fluctuations even though the RI of the pure water was constant. Since those rapid fluctuations were synchronized with the operation of the peristaltic pump, they are caused by disturbance from the water flow when the samples were exchanged. In contrast, δfrep1, in the active sensing OFC, exhibited a combination of a step-like change with the sample RI and the slow drift shown by δfrep2. This combination of behaviour in δfrep1 is detrimental to the RI sensing performance in the single sensing OFC configuration.
Figure 5b shows a sensorgram of δ∆frep calculated by subtracting the green line (δfrep2) from the blue line (δfrep1) in Fig. 5a. The temperature drift almost disappeared, and only the step-like change with the sample RI was present in δ∆frep. The mean and the standard deviation of δ∆frep were 0.76 ± 0.19 Hz at 0 vol% or 1.3334 RIU, -7.58 ± 0.24 Hz at 1 vol% or 1.3350 RIU, -16.48 ± 0.52 Hz at 2 vol% or 1.3366 RIU, -25.64 ± 0.53 Hz at 3 vol% or 1.3382 RIU, -34.59 ± 0.31 Hz at 4 vol% or 1.3398 RIU, and − 43.12 ± 0.34 Hz at 5 vol% or 1.3414 RIU, as shown by red plots in Fig. 5c. From these values, we calculated a relationship between the sample RI and δ∆frep, indicated by the red circles in Fig. 5c. The linear relationship between the sample RI and δ∆frep was obtained with a correlation coefficient (R) of 0.9935. The good fitting result indicated that the dual-comb effect minimizes the effect of temperature fluctuations.
Rapid Detection Of Sars-cov-2 N Protein Antigen
Antibody modification of the intracavity MMI fibre sensor creates a photonic RF biosensor for the detection of target antigens through antibody-antigen reactions because the RI-dependent frep shift is converted into an antigen-concentration-dependent frep shift (see Fig. 1a). We confirmed the effectiveness of the active-dummy compensation with the dual-comb configuration in high-precision RI sensing demonstrated above. The resulting enhanced RI precision covers the effective RI change expected by the antigen-antibody interaction on the sensor surface, enabling us to apply these dual sensing OFCs for rapid, high-sensitive detection of viruses/pathogens and biological molecules.
The concept of the antigen-antibody interaction (in this case, a viral protein) was applied for the detection of SARS-CoV-2 protein with dual-comb biosensing. Among several proteins in SARS-CoV-2, the N protein, which functions to package the viral RNA genome within the viral envelope into a ribonucleoprotein complex, is a promising candidate for antigen-antibody interactions because of its abundance, low probability of mutation, and relatively low molecular weight. Thus, we used the combination of a commercialized N protein monoclonal antibody (Fapon Biotech Inc., Dongguan, Guangdong, China, FPZ0553) and a commercialized N protein recombinant antigen (Fapon Biotech Inc., Dongguan, Guangdong, China, FPZ0513) for the antigen-antibody interaction for the intracavity MMI biosensor (see Fig. 1b), exhibiting high affinity in enzyme-linked immunosorbent assay (ELISA). We made a surface modification of the MMI fibre sensor (material = SiO2) with amino-terminated groups through a silane coupling reaction for a self-assembled monolayer (SAM) after surface cleaning and modifying by UV ozone. Then, the N protein antigen was immobilized on the amino-group-coated MMF fibre sensor surface to realize an active sensing OFC. Additionally, the SAM without immobilized antibody was applied to the surface of the MMI fibre sensor for the dummy sensing OFC. The MMI fibre sensors with and without surface modification by the immobilized antibody were placed together in the same sample cell for the active and dummy sensing OFCs, respectively (see Table 1 and Fig. 3d). Solution samples of the N protein antigen in phosphate-buffered saline (PBS) at different molar concentrations were consecutively introduced into the sample cell with a peristaltic pump. The N protein antigen-antibody interaction could only occur on the sensor surface of the active sensing OFC because that of the dummy sensing OFC did not include immobilized antibodies. Thus, the dummy sensing OFC was used for a negative control here.
Figure 6a shows the sensorgram of δfrep1 and δfrep2 as the molar concentration of the antigen/PBS solution increased from 1 aM (blue zone), 1 fM (green zone), 1 pM (yellow zone), and 1 nM (red zone) after starting with pure PBS (purple zone); this range of molar concentrations was selected for the sample considering the LOD for SARS-CoV-2 of RT-PCR. The time period for data acquisition (see colour-highlighted zones in Fig. 6a) was set to 10 min. In the grey zones (time period = 8 min), we performed the following three steps: (1) we introduced the antigen/PBS solution into the sample cell with the peristaltic pump (1.5 min), (2) we waited for the antigen-antibody interaction to occur (5 min), and (3) we rinsed the sensor surface with PBS to flush the N protein antigen that did not interact with the antibodies away (1.5 min). Since the step-like change in δfrep1 with the antigen concentration was completely overshadowed by the background temperature drift, we calculated the frequency difference (δ∆frep) between δfrep1 and δfrep2 to eliminate the influence of temperature drift as described in the previous subsection. Figure 6b shows the sensorgram of δ∆frep. Focusing on the zones highlighted in colours other than grey, a slightly dull stepped change in δ∆frep dependent on the molar concentration was observed, although a small drift in δ∆frep within the range of a few Hz remained at each molar concentration. To evaluate the validity of this behaviour in the sensorgram, we calculated the relationship between the molar concentration and δ∆frep, as shown by the red circles plotted in Fig. 6c. The negative slope was consistent with the RI dependence of δ∆frep (see the red line plotted in Fig. 5c) because the progression of the antigen-antibody reaction increases the effective RI near the MMI fibre sensor and hence decreases frep [38]. In this way, we demonstrated the potential for rapid detection of the SARS-CoV-2 N protein antigen within this range of molar concentrations.