*Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells DOI: http://dx.doi.org/10.5772/intechopen.94248*

#### *2.1.1 Surface plasmon resonance (SPR)*

SPR is a sensitive label-free technique for characterization of molecular interaction and detection of molecules through the affinity recognition. Due to surface plasmons excited on the surface of a thin metal film and an evanescence field created on surface, the refractive index change in the vicinity of the sensor surface (within ~200 nm) can be detected. This relatively large sensing depth allows SPR to monitor the changes occurred not only on surfaces, but also in the bulk. SPR has been applied in various studies for molecules with molecular weight between 1000 Da~500 kDa [47]. Liu et al. [48] attached human ovarian carcinoma SKOV-3 cells on the ceiling of a flow cell chamber, and detected their vascular endothelial growth factor (VEGF) secretion by monitoring the SPR signal of the antibodiesimmobilized gold chip at the bottom of the flow cell. This SPR sensor has a linear dynamic range of 0.1–2.5 μg mL−1. Of note, even though SPR features label-free measurement and high sensitivity, the lack of spatial resolution restricts its application in the mapping of protein secretion. In the meantime, SPR imaging is suitable to achieve this goal. Instead of measurement of the SPR angle shifts in SPR spectroscopy, plenty of SPR imaging systems use fixed angle and wavelength of incident light for the excitation of SPR so that the reflection intensities of multiple spots within imaging area are obtained simultaneous.

Milgram et al. [49] took advantage of SPR imaging to real-time monitor the secretion of immunoglobulins from B-cells hybridoma. They assembled an antigen microarray using electro-copolymerization of free pyrrole and pyrrole-modified antigen on a gold chip. The secreted immunoglobulins were captured by the antigen proteins, triggering the refractive index changes. The SPR intensity changes were consequently observed at a fixed angle using a 12-bit CCD. A sharp SPR kinetic curve was observed after several minutes of incubation, indicating fast and sensitive detection of immunoglobulins. Wu et al. [50] used anti-CD4 antibody to capture and immobilize human CD4+ T-cells on a sensing chip, and detected their in-situ secretion of Interferon gamma (IFN-γ) through functionalized anti-IFN-γ antibody located at the neighboring sites. The detection limit for IFN-γ was ~50 ng/mL. Stojanović et al. [51] applied SPR imaging to quantify the antibody production from single EpCAM hybridoma cells. Based on the measured SPR signal alteration, the overall secretion antibody amount from a single cell was calculated as 0.02 to 1.19 pg per cell per hour. However, this estimation is questionable because not all the antibodies secreted from cell were captured. To solve this problem, they performed simulation using COMSOL Multiphysics and found the captured antibodies by sensors accounts for 99% of the excreted antibodies, only 1% excreted antibodies are not detected [52].

A major limitation of intensity-based SPR imaging is its relatively low sensitivity. Compared to SPR of high sensitivity, intensity-based SPR imaging suffers from one order of magnitude lower of sensitivity [53]. This relatively low sensitivity is dominantly contributed by the fluctuations of the incident light intensity, photon statistics associated shot noise, and detector noise [54]. This issue could be improved by performing optical multilayer structured long-rang surface plasmons [55], angle-resolved or spectral SPR imaging [56], NIR light source [57], etc.

#### *2.1.2 Nanoplasmonic biosensors*

Different from SPR sensors utilizing propagating surface plasmons generated on flat metal film, nanoplasmonic sensors generate and manipulate localized surface plasmons on nanostructures [58]. Conventionally, nanoapertures or nanoparticles are fabricated to interact with light, leading to localized surface plasmon resonance

*Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells DOI: http://dx.doi.org/10.5772/intechopen.94248*

(LSPR) or extraordinary optical transmission (EOT). As nanoplasmonic biosensors combine with cell imaging, a powerful tool is created for protein secretion imaging. Nanoplasmonic biosensors generate strong evanescence field in the vicinity of less than 30 nm, thus are highly sensitive to the local refractive index changes, allowing the detection of target molecules captured on surfaces. Since the excitation of these nanostructures did not require complex optical devices, the instrumentation can be simple and straightforward. By tracing the dynamic alteration of reflection or transmission spectra, these changes can be recorded in a simple and real-time manner. Therefore, this feature allows a simple-but-sensitive label-free detection.

#### *2.1.2.1 Localized surface plasmon resonance (LSRP)*

LSRP generated on nanostructures results in the collective oscillation of electrons at the interface of metallic structures. A localized electromagnetic field is sensitive to the nanostructure shape and changes of refractive index at the distance of 10–30 nm [59, 60]. A molecular binding event on these nanostructures, causes a red shift in SPR peak, and gives rise to the scattering intensity of light at the same time. Due to a short electromagnetic field decay length, LSPR is insensitive to the changes of the refractive index in bulk, therefore the bulk effect can be minimized. This feature allows LSPR to measure temperature-dependent binding process, and investigate the effects of various environment factors on molecular interactions, such as solution pH and ionic strength [61].

LSPR measurement can be implemented by recording either SPR peak shift or scattering intensity changes. In the SPR peak shift based detection, Zhu et al. [62] combined microwell technique with LSPR to monitor Interleukin 6 (IL-6) secretion in the single cell level. The microwells were adapted to trap cells, a gold-capped nanopillar-structured cyclo-olefin-polymer film was covered on the top of microwells. The transmittance spectrum of the gold nanostructured surface provides real-time information on the absorption peak shift of nanogold during cell secretion. This nanostructured film further fabricated with Anti-IL-6 antibody realized a detection limitation of 10 ng/mL for IL-6.

In the scattering intensity based detection, Oh et al. [63] developed a multiplexed LSPR system for simultaneous measurements of pro-inflammatory cytokines (IL-2, IFN-γ, and TNF-α) and anti-inflammatory cytokines (IL-10) secreted by T cells. The cell culture and cytokine detection were conducted in independent steps. The sensitivity reached 20−30 pg/mL. Faridi et al. [64] applied similar system to characterize the secretion of IL-6 and tumor necrosis factor alpha (TNF), secreted from human monocytes and lymphocytes. The same principle has been applied to monitor neutrophil extracellular traps (NETs) and fibril released from single neutrophils [65]. Raphael et al. [66] achieved the real-time imaging of anti-c-myc antibodies secreted from single hybridoma cells with gold nanostructured arrays. The electron beam lithography was implemented to fabricate the square arrays of gold nanostructure on glass coverslips. The gold nanostructure arrays have a diameter of 70 nm, a height of 75 nm and a separation distance of 300 nm. The differing distances between the position of the settled single cell and the centers of arrays provide spatial resolution to observe the protein secretion. The c-myc peptides conjugated to plasmonic gold nanostructures captured the secreted anti-c-myc antibodies in a real-time manner. The caused binding displayed an increase in scattering intensity due to LSPR effect, which is measured through changes of light reflection. In parallel with reflected light based LSPR characterization, the transmitted light and fluorescence microscopy were integrated for live cell imaging. The transmitted light imaging enables observation of the position of single cell and its morphological change. The fluorescence microscopy allows

the monitoring of membrane dynamics through a cell plasma membrane labeldye rhodamine DHPE, which distinguishes the signal due to occasional outward protrusions of lamellipodia from protein secretion signal. To model the singular cell secretion, the cell was assumed a spherical emitter producing a propagating pulse of antibodies with a Gaussian concentration profile, where the diffusion constant D = r 2 /6•t. This method provides a sensitivity of 100 pM (~4.88 ng/mL) for the detection of anti-c-myc antibodies.

### *2.1.2.2 Extraordinary optical transmission (EOT)*

In EOT biosensor, regularly periodic nanohole structure of subwavelength in a metallic film results in significant enhancement of light transmission through the nanoholes. This phenomenon is associated with both localized and propagating surface plasmons on the nanohole structures. By collecting and analyzing the transmission spectra, the light frequency-dependent transmission enhancement could be easily recognized. The molecular binding event on surface is corresponding to the transmission spectral shift. Consequently, the binding event can be visualized in a real-time manner through tracking the changes of transmission spectra. Combining localized and propagating surface plasmons, the spectra of EOT provide a wealth of information with varying sensitivities at different regions of nanoholes. In addition, the nanohole structures allows the flow-through design, which changes the manner of the mass transfer of analytes, and increases delivering rate of analytes from bulk to the sensing surface [67].

To achieve real-time imaging of cell secretion, Li et al. [68] first developed a microfluidic device that separated a cell culture module and an EOT sensing module. The nanohole array sensor has a hole diameter of 200 nm and a periodicity of 600 nm, and was fabricated by deep-UV lithography and functionalized by biotinylated antibodies. This detection module is directly connected with an adjacent cell culture module made of a zigzag single-channel PDMS unit. This biosensor system achieved a detection limit of 145 pg/mL for VEGF. In this study, the VEGF secretion was detected from the media containing a group of cancer cells, and the mapping of secretion was disabled due to the separated configuration of detection and cell culture modules. Subsequently, they developed a microfluidic system suitable for the secretion imaging of a single cell [69]. Enclosed in a microchamber, a single cell was attached to nanohole arrays, functionalized by antibodies. To achieve the function of real-time imaging, spectrum profiles on a perpendicular 1D line was collected, and the selected positions of the region-of-interest were analyzed. This sensor achieved a detection limit of 39 pg/mL for interleukin-2 (IL-2) in complex media.

#### **2.2 Photonic Crystal Resonator (PCR)**

Similar with plasmonic sensors, Photonic Crystal Resonator (PCR) exploits an evanescence field to interact with and sense the surrounding medium, i.e. the changes of its refractive index. PCR is created by Photonic Crystals (PCs) that own periodically varied refractive indices, which forbid the light propagation of certain wavelength of light in certain directions inside the material. This causes constructive or destructive interference of the light, and therefore a minimum in the transmission spectrum and a maximum in the reflection spectrum could be observed. Dependent on material, geometry and the index contrast, the decay length of PCs ranges from dozens of nanometers to the order of a few microns [70]. PCs composed of silicon nitride (Si3N4) or titanium dioxide (TiO2) have been proved able to achieve cellular imaging with resolution 2–6 μm [71].

#### *Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells DOI: http://dx.doi.org/10.5772/intechopen.94248*

Juan-Colás et al. [72] monitored the secretion of thrombopoietin at single cell level using photonic crystal resonant imaging (**Figure 2**). The PCR surface microfabricated by electron beam lithography, consists of a grating with period of 555 nm on a 150-nm-thick Si3N4 film. It displayed a penetration depth of ~200 nm. To realize hyperspectral resonance image, reflection spectra was taken in a sequence of illumination wavelengths with 0.25-nm wavelength step. Under each wavelength, the intensity value of each pixel was analyzed and fit into a Fano resonance curve to accurately obtain the resonance wavelength for each pixel. To create a sensitive surface for analyte capture, antibodies were functionalized on a 3D matrix consisting of branched glucan dextran, which increased the density of antibodies on the surface. This sensor was demonstrated to detect lower than 125 ng/mL of suspended recombinant Human thrombopoietin. The interaction of antibodyantigen was treated as a single adsorption process, and the protein secretion was modeled as a Langmuir adsorption distribution. By quantifying the total secretion area in a timely manner, the single-cell secretion rate was calculated as 22 μm2/h. In addition, the wide field of view allows parallel imaging of 30 cells on an area of 500 × 500 μm, so that their dynamics and the kinetics could be characterized simultaneously.

#### **Figure 2.**

*Photonic crystal resonant imaging protocol for monitoring TPO secretion from a single BHK-TPO cell. (a) Dextran molecules are employed to create a 3D network of antibodies. Casein protein is employed as a blocking agent to prevent nonspecific binding from other proteins expressed by the cells. The secreted cytokines (i.e., TPO) from attached cells diffuse through the 3D network to specifically bind to the antibodies immobilized throughout the dextran network. The amount of deposited casein protein is optimized to maximize the signal-to-noise ratio of the detection system. (b) The region of interest (55 × 40* μ*m), with a wavelength uniformity of* Δλ *± 0.5 nm. (c) A hyperspectral PCRS image reveals the adhesion of a -BHK-TPO cell to the PCRS, whose high concentration of cell adhesion molecules located in the inner region is translated into a higher refractive index content area. (d) Over time, this secretion area increases as TPO molecules are secreted from the cell and bind to the surface-immobilized antibodies, therefore locally increasing the refractive index around the BHK- TPO area. (e) The secretion of TPO is then monitored over time, and a Langmuir adsorption distribution is fitted to the data to model the secretion from the BHK-TPO cell accounting for the area covered by the adhered cell. Reprint (adapted) with permission from Ref. [27] under the terms of the Creative Commons Attribution License. Copyright 2018 Juan-Colás et al.*

#### **2.3 Interferometric Scattering Microscopy (iSCAT)**

Interferometric Scattering Microscopy (iSCAT) is a single-molecule detection based approach. It relies on the light scattered by subwavelength objects. The signals come from the interference between the scattered light by the detected object and a reference light (**Figure 3d**). With the capability of single-molecule detection, iSCAT has shown its remarkably high sensitivity in cell imaging, singleparticle tracking, label-free imaging of nanoscopic (dis)assembly, and quantitative single-molecule characterization [73]. Meanwhile, iSCAT microscopy itself does not provide adequate chemical or biological specificity due to its nature in collection of scattered light from all small objects [74].

McDonald et al. [75] reported an iSCAT contrast method to distinguish proteins secreted from an Epstein−Barr virus (EBV)-transformed B cell line (**Figure 3a**). The observed contrast on an iSCAT image reflects the amplitude of the electromagnetic field scattered by proteins, which is directedly correlated with the scattering cross-sections of detected molecules (**Figure 3c**). Here, the iSCAT demonstrated its capability of monitoring secreted proteins with varying molecular

#### **Figure 3.**

*Secretome quantification and identification. (a) Cartoon of the detection region. (b) Histogram of detected proteins during a 125 s long measurement period in which detected contrasts are counted in bins of 1 × 10–4 contrast (diagonal-hatched blue bars). 503 distinct proteins were counted in this period. The red arrow indicates the expected contrast corresponding to an IgG dimer. Superimposed over the cell secretion data is the contrast distribution of a purified IgG solution injected into the iSCAT FOV with a micropipette (diagonal-hatched rose bars, normalized for clarity). The detected secretion events from a single Laz388 cell on an anti-human IgGfunctionalized coverglass are also shown (125 s integration, hollow black bars). The anti-human IgG surface selectively binds all four IgG antibody subtypes and resists the adsorption of other proteins. (c) Comparison of 2.5 fps (400 ms) and 25 fps (40 ms) image acquisitions of a single Laz388 cell's secretions. The right column shows three sequential 40 ms images, while the left displays the 400 ms image composed of 10 total images, including the three shown on the right. Scale bar: 1* μ*m. (d) IgG secretion rates and secretion heterogeneity. Reprinted (adapted) with permission from Ref. [28]. Copyright 2018 American Chemical Society.*

*Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells DOI: http://dx.doi.org/10.5772/intechopen.94248*

weight 100 kDa−1 MDa. To provide iSCAT the detection specificity for human Immunoglobulin G(IgG), the sensing surface was functionalized with anti-human IgG, which showed high specificity on the adsorption event of IgG (**Figure 3b, d**). The quantification of IgG was realized by counting over certain integration period. It was found that the secretion of IgG antibodies in single Laz388 cells proceeded at a rate of 100 molecules per second.

One of the challenges in iSCAT for cell secretion is that, the recognition of small molecules is difficult due to their limited iSCAT contrast. In above work, the minimum protein molecular weight from iSCAT was approximately 100 kDa. Most cytokines have molecular weight range from approximately 6 to 70 kD, which fall out of the iSCAT's detection range.
