Plasmonic random laser biosensor on fiber facet for label-free detecting biomolecules

: Low-cost and miniaturized biosensors are key factors leading to the possibility of portable and integrated biomedical system, which play an important role in clinical medicine and life sciences. Random lasers with simple structures provide opportunities for detecting biomolecules. Here, a low-cost biosensors on fiber facet for label-free detecting biomolecules is demonstrated resorting to plasmonic random laser. The random laser is achieved resorting to a random plasmonic scattering structure of Ag nanoparticles and polymer film on fiber facet. Refractive index sensitivity and near-surface sensitivity of the biosensor are systematically studied. Furthermore, the biosensor is used to detect lgG through specific binding to protein A, exhibiting the detecting limit of 0.68 nM. It is believed that this work may promote the applications of plasmonic random laser bio-probe in portable or integrated medical diagnostic platforms, and provide fundamental understanding for the life science.


Background
The growing requirements of clinical medicine and life sciences spark off a remarkable trend of bio-sensing technology 1,2 , which provide important insight into pharmacological intervention and cellular processes 3 . Optical biosensor is one of the most widely used biosensors [4][5][6] . It commonly obtains the information of target biomolecules through detecting changes in optical signals that caused by the molecule interaction 7 . Towards the advanced sensing technology, optical biosensor initially employs labeled biomolecule to extract information about target biomolecules through the spectral characteristics (eg. fluorescence intensity variation or wavelength shift) 8,9 . However, label-based biosensor would weaken the biological activity or interfere the biological interaction. Label-free biosensor without biomarkers effectively avoid these negative factors from labelling process, providing new functionalities and opportunities 10 . As is well known, high sensitive and detection limit play a central role of sensors 11 . Therefore, great efforts are needed to develop new types of label-free biosensor to optimize the sensing performance.
Laser with narrow spectral linewidth and high intensity implemented as label-free biosensors could resolve smaller wavelength shifts, which is associated with a smaller vitiation of analytes 12 . Biological molecules attached to dielectric cavity can change its effective refractive index, leading to the changes of resonant mode. Currently, laser-based label-free biosensors with extremely high detection sensitivity have been achieved resorting to different kinds of cavities, such as whispering gallery mode lasers 13 , distributed feedback lasers 14,15 , photonic crystal lasers 16,17 and plasmonic lasers 18 . However, label-free biosensing by traditional lasers suffers severely from meticulous designs and precise preparations of the special optical resonator cavities, which are challenging and high-cost for sensing. Therefore, it is extremely desirable to design label-free laser biosensor with characteristics of simplified preparation and low-cost to promote the development of portable and integrated biomedical system. Random lasers (RL) characterized with simple structure and flexible design, are good candidates for label-free laser biosensor 19,20 . Without rigid and expensive optical cavities, random laser is based on the multiple scattering of light in low-cost disorder system. The lasing characters are highly dependent on the gain 21,22 and scattering structure 23 24 . Based on this feature, such random lasing sensors have been used to sense of temperature 25 , chemicals 26 , tumor tissue 27 , cell counting 28 , and bone tissue structure 29 . Furthermore, random laser sensors based on optical fiber are proposed 30 , which not only retain the advantages of the random laser sensor, but also make full use of the high optical transmission efficiency of the optical fiber. The compact structure promotes the miniaturization and integration of the random laser biosensors, bringing new opportunities for portable and integrated biomedical sensing system. However, random lasers biosensors based on optical fiber for monitoring biomolecules interactions have not been investigated till now. In view of their potential functions for life sciences and medical diagnosis, biomolecules sensors based on random laser with optical fibers are in a great need to be developed.
Here, a label-free biosensor based on low-cost plasmonic random laser on fiber facet is designed, fabricated, and demonstrated. The random lasers are fabricated by covering polymer membrane over the self-assembly silver nanoparticles (Ag NPs) randomly distributed on fiber facet. We have systematically studied the random lasing action and explored their applications in refractive index sensing and near-surface sensitivity, revealing the important feature that the wavelength shift of lasing mode is proportional to the changes in external refractive index of random laser. The biosensing capability of the random laser on fiber facet is demonstrated by detecting human lgG. Through specific binding to protein-A fixed on the surface of random laser, the concentration of lgG has been well monitored.

Results and discussion
In our experiment, Ag NPs with local surface plasmon resonance act as the scattering structure of the random laser, which are self-assembled on the fiber facet by polyvinylpyrrolidone (PVP)-assisted reaction 26 , as is shown in Figure 1a  The mean diameter of Ag NPs is estimated as 25 nm. The extinction spectrum exhibits a broad peak from 350 nm to 650 nm (see in Figure 1d). The normalized electric field distribution near Ag NPs is stimulated. Due to the localized surface plasmon resonance (LSPR) of Ag NPs, their local fields are 10 times enhanced (inset of Figure 1d) respected to air, which can enhance the emissions from the random lasers 20,31-33 . Poly [9, 9-dioctylfluorenyl-2, 7-diyl] end capped with DMP (PFO) is an attractive light emitting polymer, characterized with high luminescence efficiency and high charge carrier mobility 34 . Additionally, PFO film with negatively charged surface has been applied in chemical sensors, biological labels and optoelectronic device [35][36][37] .
Thus, PFO chosen as the gain material for the random laser, is transplanted to the fiber facet ( Figure 2a). Optical micrograph of the front view of the random laser on fiber facet illustrates that the surface of the polymer is uniform and smooth (inset of Figure 2a). The absorption spectrum of PFO displays a absorption band from 320 nm to 430 nm, which is overlapped with the extinction band of Ag NPs (blue shading) ( Figure 2b). It indicates that the excitation of random laser could be enhanced by Ag NPs 38 . The photoluminescence spectra of PFO with or without Ag NPs are shown in Figure 2b, respectively. These two photoluminescence spectra demonstrate that the fluorescence intensity is enhanced by Ag NPs. And the photoluminescence lifetime is further studied by comparing the photoluminescence decay dynamics of PFO with (red dots) and without Ag NPs (blue dots). It can be confirmed that the interaction with Ag NPs reduced the photoluminescence lifetime from 0.8 ns to 0.5 ns ( Figure   2c) 20 . In addition, the local field enhancement by Ag NPs has been also been demonstrated by simulating the normalized electric field distribution near Ag NPs that randomly distributed on fiber facet. The result in Figure 2d demonstrates that Ag NPs, especially the Ag NPs dimmer with gap of 5-10 nm, could provide enormous local enhancement and enhance the scattering light from Ag NPs [39][40][41][42] . As a result, the Ag NPs can supply strong gain and effective feedback for the random lasing. The emission characteristic of the plasmonic random laser on fiber facet is systematically investigated. Figure 3a shows the schematic of the experimental setup.
The random laser is directly pumped by a pulse laser at 400 nm. The emission is detected at angle θ, which is respect to the orientation vertical to the fiber. Figure 3b depicts the evolution of the emission spectra by varying the pump energy densities.
When the pump energy density is 19 μJ/cm 2 (red curve in Figure 3b), the spectrum exhibits a broad spontaneous emission band (black curve in Figure 3b). By increasing the pump energy density to 33 μJ/cm 2 , the spectrum appears a narrow emission band centered at λ = 468 nm with several sharp spikes. It should be noticed that the linewidth of the sharp peaks recorded by a high-resolution spectrometer is as narrow as 0.42 nm (Figure 3c), indicating the coherent feedback being formed by Ag NPs. As the pump energy density further increases to 39 μJ/cm 2 and 60 μJ/cm 2 , the emission intensity increases more rapidly (blue and green curves shown in Figure 3b). Figure   3b plots the variation of emission intensity at 468 nm as a function of the pump energy density. There is a clear threshold behavior with a knee point in the curve as the pump energy densities increase, which further suggests the occurrence of random lasing. According to the threshold curve, the threshold of random lasing is measured at 30.5 μJ/cm 2 . The photograph inset of Figure 3d shows the bright optical fiber excited by the pump beam on the end of fiber, demonstrating the random laser operating well. Furthermore, spatial emission performance of this plasmonic random laser on fiber facet is studied by changing detection angle from 5º to 175º under a fixed pump condition. The integrated intensities of observed spectra in Figure 3e increase from the sides to the center. As the thickness of the PFO film decreases from the central to the edge of the fiber facet during the film-forming process, the random laser performs an optimal spatial distribution 43 .
where n is the effective refractive index of gain, L is the resonant cavity length, m is a positive integer mode number, and λ is the lasing wavelength. Therefore, the media with different refractive index that exposed to the RL surface would affect the resonant mode by altering the effective refractive index of gain. Figure   The excellent surface sensing behavior of the random lasers makes it possible to detect biomolecules based on surface adsorption 46 . Figure 5a shows the sensing process for the RL on fiber facet based on a protein-protein interaction 14 . An initial baseline of emission wavelength is established before functionalizing the surface (the red line in Figure 5b). Protein A is firstly adsorbed to the surface of seven random lasers through noncovalent hydrophobic attachment for 20 min at room temperature.
Then the fiber facet is subsequently rinsed by phosphate buffered saline (PBS).
Corresponding antibody (human IgG) solutions with the concentrations of 34 μM is exposed to the surface of RL decorated by Protein A. The human IgG molecules are specifically binding to Protein A by immunization interaction. The corresponding sensing kinetics for human IgG at 34 μM with spectra measured every 5 min. As shown in Figure 5c, the wavelength red-shift tends to stable after 15 min, indicates that the sensing system reaches dynamic equilibrium during the binding process.

Conclusions
In summery, a label-free RL biosensor on fiber facet is achieved based on protein-protein interaction. Plasmonic random scattering structure of Ag NPs is simply self-assembled on the facet of optical fiber by a hydrothermal method. The random laser on fiber facet is prepared by transplanting the PFO membrane upon the scattering structure, which operates with a low threshold of 30.5 μJ/cm 2  A parameter θ is introduced to characterize the detected direction with respect to the orientation vertical to the fiber.

Sensitivity measured of a Protein Monolayer
Poly (Lys, Phe) (PPL, Sigma-Aldrich) is dissolved in DI water with concentration of 1 mg/ml. After being rinsed by PBS (pH=7.4) solution, the plasmonic random laser on fiber facet is exposed to the PPL solution. The PPL monolayer gradually self-assembled on the surface of RL. The emission spectra for different cultivating time are recorded. The wavelength shift response is saturated after 15 nm, resulting to a sensitive thickness of 50 nm.

Funding
The authors acknowledge the financial support of the National Natural Science

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Fourth Military Medical University.

Consent for publication
All the authors have approved the manuscript and agree with submission to this journal.