Super-sensitivity in label-free protein sensing using a nanoslot nanolaser

Microphotonic sensors have been actively studied with increasing demands for label-free biosensing in medical diagnoses and life sciences. For high-throughput and low-cost sensing, a high sensitivity is crucial for eliminating the pre-concentration process, while a simple setup of sensors is also desirable. This paper demonstrates a super-sensitivity for protein, which satisfies these requirements. The key device is a photonic crystal nanolaser, in particular with a nanoslot. Even using a simple setup, the nanolaser achieves an extraordinary-low detection limit for BSA protein, i.e. 255 fM on an average, which cannot be explained by its bulk index sensitivity. The specific adsorption of the protein is observed only around the nanoslot with strong laser intensity. 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Introduction
In the field of biosensing, the detection of protein is becoming important for tumor markers, allergy testing, cytoscopy and proteomics [1,2].Current methods, however, require complicated screening and concentration processes prior to sensing.Direct detection is more desirable for high-throughput analyses, but high selectivity and high sensitivity giving a low detection limit (DL) (pM order for tumor markers [3]) are required.The selectivity is mainly attributed to antigen-antibody reactions and surface chemistry, while the sensitivity is attributed to the performance of sensors as transducers.The sensor chip and the overall system must be simple for cost reduction and disposable use, and the chip should be small enough to integrate with advanced inspection kits such as µ-TAS.Now, we focus on the sensitivity of sensors as transducers.Fluorescent labels, which achieve a low DL of pM order [4,5], are widely used as transducers in biosensing.However, they are suspected to denature the targets and their functionalization complicates the overall process.Therefore, label-free methods using surface plasmon resonance (SPR) [6] and passive optical cavities [7][8][9] have been studied as alternatives.They detect the adsorption of biomolecules through a resonant wavelength shift ∆λ s (or resonant angle).In general, however, they have issues such as large size, low sensitivity, wide resonant spectrum (linewidth ∆λ w ), and/or complicated setup.Being proportional to ∆λ w /∆λ s , DL has been limited to nM regime in most cases; a lower regime is achieved only by using an ultrahigh Q cavity with complicated equipment [8], which may not be low-cost or disposable.
Recently, nanolasers have also been studied for sensing [10][11][12][13][14][15][16].They are photopumped and the emission is detected through the same free space optics, hence they operate remotely without the need for any electrical contacts.Cost reduction, disposable use and integration are possible when the nanolaser as a sensor chip is isolated from other equipment.A small size and low DL can be achieved simultaneously, because ∆λ w under lasing is reduced independently of the Q factor when the spectral broadening due to thermal chirping [11] is suppressed.As a device specifically suitable for this purpose, we have reported the nanoslot (NS) photonic crystal nanolaser [12], as shown in Fig. 1.In high-index-contrast waveguides and cavities, the modal electric field is confined strongly in the NS [17], resulting in enhanced light-matter interaction and high sensitivity in liquids [12,18].Furthermore in water, our NS nanolaser has low thermal chirping and is particularly effective for a narrow ∆λ w of 18 pm with a high sensitivity of 410 nm/RIU, giving a liquid index resolution of 4.5 × 10 −5 [12].In this paper, we discuss protein sensing using this NS nanolaser and show that the advantage of the NS is not limited to the narrow spectrum.In this experiment, it exhibits a super-sensitivity giving a remarkably low DL.It is confirmed to originate from the accelerated adsorption of the protein at the NS, which may be occurring due to optical trapping.In the following, we briefly describe the device structure, fabrication, and the procedure of sensing a standard protein-bovine serum albumin (BSA).We then show the details of the sensing characteristics, particularly focusing on the unique behaviors in the regime of ultra-low BSA concentration.We present simple theoretical analyses and discuss the advantage of this device compared to others.

Device structure and protein adsorption process
As illustrated in Fig. 1, two airholes in a triangular-lattice air-bridge photonic crystal slab are shifted outwards (H0 structure [11,13,19]), and a NS with width w NS is perforated.The laser mode is localized inside and around the NS.The laser wavelength shifts with the surrounding index n.The detailed device structure and the sensitivity against liquids, ∆λ s /∆n, have been reported in [12].Devices with w NS = 30−70 nm are fabricated into commercial GaInAsP/InP quantum-well wafer by using e-beam lithography, HI inductively-coupled plasma etching and HCl wet etching.Since the footprint of a single device is only ~10 µm square, highthroughput fabrication is possible even using e-beam lithography.The device is operated by pulsed photopumping at 980 nm (duty-ratio of 200, spot diameter of 20 µm) to avoid the excess heating and the subsequent laser emission is detected using a fluorescence microscope setup.The device does not show any degradation even after operating for 8 hours in water.Since the single sensing was completed within 1 minute in this experiment, the device does not deteriorate at all after hundreds of measurements.
Figure 2(a) shows the procedure of BSA adsorption.First, we functionalized the device with a hydroxyl group with 9% HCl / deionized water at 3°C, then with an amino group with 10% N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (APTES) and an aldehyde group with 2.5% glutaraldehyde (GA) at 20°C.We used BSA (molecular weight = 68 kDa) [20] as a target protein which nonspecifically cross-links with GA. Figure 2(b) shows scanning electron micrograph (SEM) and atomic force micrograph (AFM) around the NS after soaking in highconcentration BSA aqueous solution of 10 µM for more than 30 minutes.More than 10-nm roughness is observed on the surface and in the NS, indicating the BSA adsorption, which was not observed before the process.Since BSA is known to have an elliptical shape and a molecular size larger than ~3 nm × 8 nm [21], the larger roughness observed suggests that the high-concentration BSA molecules agglutinate due to hydrophobic interaction and/or crosslinking through GA.

Observation of super-sensitivity
Figure 3(a) shows laser spectra for devices in water before and after the BSA adsorption.Here, the BSA concentration was 10 µM so that the device surface is covered with BSA almost completely.The clear redshift of the spectrum appears for both devices with and without the NS.Without the NS, the spectra are broadened by thermal chirping (∆λ w > 600 pm).With the NS, the spectra are markedly narrowed due to athermalization [12] (∆λ w < 30 pm), and the resolution is drastically improved.Figure 3(b) summarizes the dependence of ∆λ s on the NS width.The data scattering does not depend on the NS width.Therefore, it is thought to be due to the nonuniform adsorption of BSA between devices.The average ∆λ s with and without the NS are 4.4 and 2.2 nm, respectively.We theoretically estimated the overlap of the laser mode with the BSA film of a constant thickness using finite-difference time-domain (FDTD) simulation (see Supplements).It predicted a 1.2-fold enhancement of ∆λ s by the localized field in the NS and could not explain the 2-fold enhancement in Fig. 3(b).These results suggest that the adsorption is accelerated by the NS and the excess adsorption occurs nonuniformly around the NS.The accelerated adsorption is also suggested in the wide range of BSA concentrations C. Figure 4(a) shows spectral shifts for different C. The large shift in the high-concentration regime of ~10 nM, Shift H , is observed for both with and without the NS.Shift H is not saturated even in the high concentration regime of over 100 µM.This might be due to the physisorption, aggregation and phase transition [22] at particularly high concentrations (this could be a reason of the data scattering in Fig. 3(b)).On the other hand, the shift in the low concentration regime, Shift L , is observed clearly only with the NS.This was observed repeatedly for all devices in different fabrication lots, but the concentration, at which Shift L started, was distributed widely from <100 fM to >100 pM for different devices.We can consider that Shift H arises from the adsorption over all other surfaces, while Shift L from the adsorption inside the NS (see Supplements), and that the different concentration for Shift L is due to the nonuniform adsorption as well as different NS width.Provided that each adsorption process occurs independently, ∆λ s is expressed as the following linear summation of three different Langmuir adsorption isotherms [23]: where K A1 and K A2 are the affinity constants of the chemisorption inside and outside of the NS, respectively, and K A3 is that of the physisorption.The latter two do not change in the presence of the NS.∆λ maxi (i = 1, 2, 3) is the maximum shift when each adsorption is saturated.The circular plots in Fig. 4(b) shows ∆λ s averaged for many devices at different C.
Error bars for the devices without the NS denote the temporal fluctuation in the spectral shift due to the broadened spectrum.For the devices with the NS, the temporal fluctuation is negligible.The solid line of Eq. ( 1) fit well with the experimental plots, when parameters in Table 1 are assumed.Here, K A2 = 1.5 × 10 7 M −1 is a standard value for BSA [20].On the other hand, K A1 is more than four orders of magnitude larger than K A2 .This means that huge enhancement in BSA adsorption, which enhances the effective sensitivity, occurs at the NS.The DL is equivalent to C at ∆λ s = ∆λ w , and is given by DLs obtained by substituting the parameters into Eq.( 2) are also presented in Table 1.The DL for the averaged plots without the NS is calculated from K A2 and ∆λ max2 to be 33 nM ( = 2.6 µg/ml, considering the molecular weight of BSA).The DL for those with the NS, calculated from K A1 and ∆λ max1 , is drastically improved to be 255 fM ( = 17 pg/ml).For the best NS device, the saturation in ∆λ s occurred at less than 1 pM, indicating that the lowest DL is less than 10 fM.Without the NS and under the strong pumping, a significant shift is not observed.With the NS, the shift increased from 0-0.1 to 0.5-0.8nm when the pump power is switched from weak to strong.These results show clearly that the adsorption of BSA is accelerated by the specific trapping at the NS [24], and it consistently explain the large enhancement of ∆λ s in Fig. 3 The trapping potential U trap is estimated directly from K Ai in Table 1 using the following equation [25]: where k B T is the thermal energy.This equation and results in Table 1 gives U trap = 9.7k B T.
Possible mechanisms of the trapping include the optical gradient force from the localized laser mode and/or from pump light, and also from the convection of water and/or thermal gradient force due to heating [26].For example, let us apply the expression for the optical gradient force [27] to the laser mode.Then, where D is the diameter of the protein when it is approximated as a sphere, n p and n w are the indices of the protein and water, respectively, ξ is the enhancement ratio of the modal energy in the NS to the average value, P is the radiated laser mode power, and V m is the modal volume in water.The highest Q in the ideal cavity is calculated to be 15,000-7,000 for w NS = 20-40 nm, respectively, also leading to V m = 0.011-0.018µm 3 and ξ = 74-46.Assuming n p = 1.5, n w = 1.321, and D = 10 nm, the trapping threshold P satisfying U trap > k B T at T = 300 K becomes 10-59 µW.On the other hand, P in the experiment is roughly estimated to be 0.01P irr = 150 µW under the strong pumping, which ensures a reasonable U trap = 15-2.5kB T in comparison with that from Eq. (3).Thus this force is a possible trapping mechanism.Other mechanisms are also worth considering, because they could explain the continuous supply of dispersed BSA molecules to the NS.

Comparison with other label-free biosensors
Table 2 compares the performance of label-free biosensors including this work.Since targets are different between reports, standard values of K A in literatures change from 10 5 -10 11 M −1 .
In general, the detection becomes easier and DL is improved for larger K A .Therefore, the performance should be evaluated from the product of DL and K A .Let us define the figure-ofmerit (FOM) factor as (DL × K A ) −1 normalized by that for the SPR sensor [28].The nanolaser achieves a FOM of 230.To date, FOMs higher than this value have been reported only for Au nanoparticles [29] and ultrahigh-Q micro-toroidal cavity [8] although they have drawbacks such as unstable DL, large sensing area, and complicated I/O.Other passive cavities and

Fig. 4 .
Fig. 4. Dependence on BSA concentration.(a) Laser spectra for different concentrations.(b) Wavelength shift with concentration.Circular plots show experimental data averaged over many devices with wNS = 30 -60 nm.Fitting curves are obtained from Eq. (1).Error bars for devices without NS denote temporal fluctuations due to the broadened spectrum.For NS devices, temporal fluctuations are negligible.

Table 1 .Figure 5
Figure5(a) shows the SEM picture at the NS after the measurement in the low-concentration regime (C < 1 pM).The specific adsorption is observed clearly under strong pumping (irradiation power at pulse peak P irr = 15 mW), and not under weak pumping (P irr = 6 mW).Figure5(b) compares spectral shifts between three different conditions for the same C. Without the NS and under the strong pumping, a significant shift is not observed.With the NS, the shift increased from 0-0.1 to 0.5-0.8nm when the pump power is switched from weak to strong.These results show clearly that the adsorption of BSA is accelerated by the specific trapping at the NS[24], and it consistently explain the large enhancement of ∆λ s in Fig.3(b) and the ultralow DL in Fig. 4(b).

Figure 5 (
Figure5(a) shows the SEM picture at the NS after the measurement in the low-concentration regime (C < 1 pM).The specific adsorption is observed clearly under strong pumping (irradiation power at pulse peak P irr = 15 mW), and not under weak pumping (P irr = 6 mW).Figure5(b) compares spectral shifts between three different conditions for the same C. Without the NS and under the strong pumping, a significant shift is not observed.With the NS, the shift increased from 0-0.1 to 0.5-0.8nm when the pump power is switched from weak to strong.These results show clearly that the adsorption of BSA is accelerated by the specific trapping at the NS[24], and it consistently explain the large enhancement of ∆λ s in Fig.3(b) and the ultralow DL in Fig. 4(b).

(Fig. 5 .
Figure5(a) shows the SEM picture at the NS after the measurement in the low-concentration regime (C < 1 pM).The specific adsorption is observed clearly under strong pumping (irradiation power at pulse peak P irr = 15 mW), and not under weak pumping (P irr = 6 mW).Figure5(b) compares spectral shifts between three different conditions for the same C. Without the NS and under the strong pumping, a significant shift is not observed.With the NS, the shift increased from 0-0.1 to 0.5-0.8nm when the pump power is switched from weak to strong.These results show clearly that the adsorption of BSA is accelerated by the specific trapping at the NS[24], and it consistently explain the large enhancement of ∆λ s in Fig.3(b) and the ultralow DL in Fig. 4(b).