346
J. Eur. Opt. Society-Rapid Publ. 22, 34( 2026)
Fig. 3. Distribution map of the specific Monkeypox virus protein and control bovine serum albumin protein molecules on the biosensor array spots. A1 – D4 shows 16 molecular binding curves recorded for all sensor spots in parallel.
Sino Biological and Sigma-Aldrich, respectively. In the biosensing experiment, MPXV proteins( 0.25 lg / lL) and control BSA proteins( 2.94 lg / lL) were immobilized onto the 4 4 gold sensor array using the following surface chemistry procedures. Initially, all gold sensor spots were incubated in an 11-mercatoundecanoic acid solution( 10.8 mM) for 28 h to produce a self-assembled monolayer. Subsequently, mixture of 1-ethyl-3-( 3-dimethylaminopropyl) carbodiimide hydrochloride( 0.4 M) and N-hydroxysuccinimide( 1 M) solution were applied to activate the sensor surfaces. It was followed by a protein immobilization process of 3 h. Finally, the sensor surface was rinsed with phosphate-buffered saline( PBS) buffer before performing MPXV neutralizing antibodies detection.
3 Results and discussion
Performance of the spectral-phase 3D SPR imaging sensor was first characterized with NaCl solutions ranged from 0 % to 15 %, which corresponding to refractive index values between 1.3330 to 1.3585 RIU [ 8 ]. Figures 2a and 2b are the experimental spectral-phase SPR images of water( 0 %, 1.3330 RIU) and 15 % NaCl solution( 1.3585 RIU), respectively. A distinct green shift is observed with increasing refractive index values. In addition, a fiber-based spectrometer( Ocean Insight, model: Ocean HR high-resolution spectrometer) was used to analyze the visible range spectral profiles. Figure 2c presents the plasmonic excitation spectra of 0 %( water), 2 %, 4 %, 6 %, 8 %, 10 % and 15 % NaCl solutions. The spectra exhibit a significant intensity increase within the green( 500 – 570 nm) and yellow( 570 – 590 nm) spectral ranges, while the red spectral region( 620 –
750 nm) is suppressed. The spectral-phase SPR images were then quantified by extracting Hue component of the HSV color space, where the Hue value represents the dominate wavelength of color [ 9 ]. Figure 2d illustrates the Hue values as a function of refractive index. It demonstrates a linear relationship within 1.3330 – 1.3501 RIU. Furthermore, slope of the sensor response curve yields a sensor sensitivity of 1191.2 Hue units / RIU.
The spectral-phase imaging biosensor was further applied for the detection of MPXV neutralizing antibodies. Figure 3 shows the distribution map of MKXV protein( red sensor spots) and the control BSA protein( yellow sensor spots) in the biosensor array. The functionalized sensor surface was initially kept at PBS, followed by the injection of MPXV neutralizing antibodies( 13 ng / lL) to facilitate MPXV protein – antibody bindings. Figure 3 presents the molecular binding curves recorded for all 16 sensor spots. On average, the injection of 13 ng / lL MPXV antibody produced a sensor response of 1.6 Hue units. In Figure 3, the discernible signals observed at sensor spots A2, B1, C1 and D2 are attributed to non-specific bindings, potentially resulting from physical adsorption of MPXV proteins. The non-specific sensor spots can serve as control sensor sites. The final sensor response for MPXV antibody was subtracted with the average non-specific sensor responses( 0.33 Hue units). In addition, the sensor measurement stability was determined to be 0.033 Hue units based on 500 measurements in PBS buffer. According to the literature [ 10 ], the biomolecular detection sensitive can be estimated to be 0.27 ng / lL using equation( 1). This value is significantly lower than the previously reported neutralizing antibodies level( 3.0 ng / lL) induced by MPXV vaccine [ 11 ].