**3. Labelling techniques**

### **3.1 Fluorescence**

Fluorescence based detection provides superb sensitivity of detection, and flexibility in labeling. Fluorescence microscopy features high spatial and temporal resolution, and capability of tracking multiple cells simultaneously [14]. Therefore, fluorescence based detection has been widely used for tracking protein secretion.

### *3.1.1 FLUOROSpot*

A simple and widely used method of single cell secretion analysis is the enzymelinked immune absorbent spot (ELISpot) that utilizes the immunosandwich-based assay for the measurement of footprint of cells [76]. Cells are loaded into wells precoated with primary antibody. Secreted proteins are captured, and further bound with labeled secondary antibodies. The addition of substrate gives rise to the spots indicative of secretion footprint. Based on similar principle, FLUOROSpot was developed which uses fluorescent dyes to replace the enzyme labels. In this way, more than one protein could be analyzed at the same time [77]. In spite that ELISpot and FlUOROSpot have been used as common methods for the detection of cell secretion, several drawbacks hinder its applications in the real-time imaging of protein secretion, including spectral overlap, varied individual spots, limited temporal resolution caused by long incubation time (12–48 h), limited number of simultaneously tracked proteins, and cell lost during the process [78].

#### *3.1.2 Microengraving*

Inspired by the principle of FLUOROSpot, the microengraving method was developed to isolate and confine single cells in a planar array of microwells. The protein secretion of segregated cells can be tracked as protein detection microarrays are integrated with the microwells, which are conventionally made of elastomeric polymers such as polydimethylsiloxane (PDMS). These microfabricated wells have subnanoliter volumes. A glass side fabricated with antibodies is covered to the array of microwells, to capture the proteins secreted by confined cells. Subsequently, the slide is removed and incubated with fluorescently labelled secondary antibodies. The single-cell cytokine secretion analysis is implemented by fluorescence imaging. Bradshaw et al. [79] showed that the same cells in the microwell can be applied on two detection microarrays successively, allowing monitoring the secretion of four types of cytokines and antibodies. In the meantime, the segregated cells in the wells can also be directly stained by immunofluorescence to determine their lineages.

A drawback in this method is that, the tracked number of proteins is limited by the capability of differentiation of multiple fluorophores within a single spectrum.

#### *3.1.3 Barcode chip*

The number of secretion protein types from above methods is limited due to the spectral overlap as multiple fluorescence labels employed on the same detection chip. This spectral overlap was overcome by separating functionalized antibodies into different lines, using a barcode design. With a proper distance between, up to 15 parallel antibody lines can be contained within a nanoliter cell-trapping chamber. This improved spatial resolution results in an enhanced its capability of simultaneous multiplex detection [28, 29, 80]. Lu et al. [81] further extended the multiplexing capacity by further improving the spectral encoding, namely, in each isolated line, three antibodies with distinct fluorescent labels are contained. This strategy enables simultaneous analysis of 42 types of proteins secreted from a single cell.

Barcode chips provide valuable information for accumulated cell secretion over a period of time. However, the underlying limitations restrict its applications on the real-time monitoring of protein secretion. For instance, the lag between cell secretion and protein detection is inevitable since capture antibody and detection antibody have to play their part in distinct steps. Given their coexistence during cell secretion, strong background fluorescence signal is expected from the unbound labeled antibodies, thus secreted proteins are difficult to be distinguished from the background noise. In addition, to remove excess probes and avoid non-specific bindings, the sensor surface after incubation with cells requires intensive wash steps, which inhibits the shortening of analysis duration into a few hours.

#### *3.1.4 Total internal reflection fluorescence microscopy (TIRFM)*

Shiraskaki et al. [82] proposed a solution for the challenge mentioned above using total internal reflection fluorescence microscopy (TIRFM) combined with microengraving method. They deposited single cells on microwells, on the bottom of which anti-cytokine capture antibodies were fabricated. Instead of separate incubation of a sensor chip with a cell and the detection antibody, the fluorescent detection antibody is present in the cell culture medium, so the cytokine capture is in step with the binding of detection antibody. An objective lens of high numerical aperture was used to achieve high incidence angles, generating evanescence field. The near-field excitation by total internal refection enhances the fluorescence signal from the detection antibody in the sandwich immunocomplex, and reduces the background signal from the unbound detection antibodies in the culture medium (**Figure 4a**). With these features, imaging of cytokine secretion was achieved within a single step (**Figure 4b**). Meanwhile, the cell staining (calcein and SYTOX) was applied to investigate the membrane integrity during IL-1b secretion (**Figure 4c**). It was found the onset of IL-1b secretion was consistent with the onset of calcein disappearance and the second protein SYTOX influx (**Figure 4d**). This phenomenon indicated the loss of membrane during IL-1b secretion. The limit of detection of this approach reached down to 2000 cytokine molecules.

#### *3.1.5 Nanoplasmonic fluorescence*

The near-field excited fluorescence can also be realized by nanoplasmonic resonator. Wang et al. [83] developed a tunable nanoplasmonic resonator (TNPR) enhanced fluorescence immunoassay for imaging of IL-2 secretion in *Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells DOI: http://dx.doi.org/10.5772/intechopen.94248*

#### **Figure 4.**

*Time-resolved monitoring of IL-1*β *secretion on the PDMS MWA chip. (a) Concept of the real-time single cell secretion assay platform. The platform works with micro-fabricated well-array chip on a fully automated fluorescence microscopy. The platform maintains the environment (temperature, concentration of CO2 and humidity) of the chip. The chip has an array of nanolitre-sized microwells with a glass bottom, into which individual cells were introduced separately. The well has open-ended structure; therefore, culture medium was exchanged constantly during the observation. The anti-cytokine capture antibody was immobilized on the well bottom, onto which secreted cytokine and fluorescently labelled detection antibody were bound to form a sandwich immunocomplex. Near-field excitation by total internal reflection enabled selective detection of the cytokine sandwich immunocomplex immediately following secretion without the requirement for wash steps. (b) Representative images of multichannel microscopy. Morphological features of a human monocyte were monitored under diascopic illumination (DIA). The fluorescence signal of SYTOX-stained nuclei was magenta (SYTOX), that of a calcein-stained cell bodies was green (Calcein) and that of secreted IL-1*β *was yellow (CF660R anti IL-1*β *Ab). Merged images of these three fluorescence signals are also displayed (Merged). Each image was obtained at the described period. Scale bar, 20* μ*m. (c) Schematic of simultaneous monitoring of IL-1*β *secretion and cell membrane integrity using calcein and SYTOX staining. SYTOX influx and fluorescent calcein disappearance was observed due to compromised plasma membrane integrity. (d) Example of the signal time course during time-resolved monitoring. Grey bands represent the period when the monocytes were exposed to ATP. Arrows represent the transition time of the respective signals. Reprinted (adapted) with permission from Ref. [31]. Copyright 2014, Springer Nature.*

submicrometer resolution (**Figure 5**). In this study, the fluorescent secondary antibody was not present while the cytokine secretion was in progress. Instead, cytokine capture and detection step was separated by washing step. With a TNPR structure of 100 nm in diameter, and an optimized fluorescence enhancement at ∼10 nm from a gold surface, the fluorescence signal was enhanced 117-fold in the TNPR area. The limit of detection was lower than 100 ng/mL.

#### *3.1.6 Bead biosensors*

Another fluorescence based strategy is associated with bead-based biosensors. The cells are confined in microwells, along with antibody-labeled microbeads, and fluorescently-labeled secondary antibody. The capture of secreted proteins on beads was accompanied by the increasing density of secondary antibody, and increasing fluorescence intensity of microbeads. A key advantage of bead biosensors is that,

#### **Figure 5.**

*Demonstration of quantitative high spatial resolution mapping of IL-2 secretion from individual Jurkat T cells. (a) Schematic of high-resolution mapping of cytokine secretion by a TNPR-enhanced in situ immunoassay. ATNPR array fabricated by NIL was incubated with anti-IL-2 antibody, and after Jurkat T cell plating, stimulation, and removal, a portion of the secreted IL-2 was captured by the antibodies on TNPR. Fluorescence-conjugated anti- bodies were applied to detect the secreted IL-2 by forming an antibody- antigen (IL-2)-antibody sandwich on the TNPR surface. Insert: SEM image of a Jurkat T cell adherent to a TNPR array (500 nm pitch). (b) quantitative mapping of Jurkat T cell secretion profiles. Reprinted (adapted) with permission from Ref. [50]. Copyright 2011 American Chemical Society.*

it breaks the limit of target-molecular adsorption kinetics, which is controlled by diffusion rates, and exists in majority of immobilized or stationary biosensors [84]. In addition, the regeneration of biosensors can be as simple as removing used beads, and infusing new beads [85]. Son et al. [86] used microcompartments as small as 20 picoliter to confine single cells, suspended antibody-labeled microbeads, and fluorescently-labeled secondary antibody together. The quantitation of secreted cytokines was achieved by tracking the intensity changes of fluorescence on microbeads. They found that the number of microbeads confined within a single microcompartment did not significantly affect the fluorescence enhancement on a single microbead. An et al. [87] utilized a microwell device for cell confinement, and detected the fluorescence changes on functionalized detection beads which are co-incubated with single cells.

Other than the design of microwell, Cui et al. [88] developed an microfluidic immunoassay device that integrated a cell culture chamber, an array of cytokine detection units and an array of active peristaltic mixers for on-chip sample mixing. Cells were isolated, cultured and biochemically stimulated in the same chamber. The detection chambers were loaded with cytometric fluorescent beads. Upon sandwich structure formed after cytokine secretion, the fluorescence intensity changes were analyzed by flow cytometry. The continuous monitoring of cytokine secretion was achieved by the extraction of a small portion of the cytokine-containing culture media to the detection chamber. With this system, the secretion profilings of IL-6, IL-8, and TNF were observed with a detection limit of 20 pg/mL and a sample volume of 160 nl.

#### *3.1.7 Cell-surface affinity sensors*

Cell-surface affinity sensors functionalize cell surface to capture secreted molecules from cells. These targets can subsequently be detected by fluorescent labeling. An appealing advantage using this strategy is that, the effect of heterogeneous spatial distribution of secreted molecules is minimized [89, 90]. As secreted molecules release from the cell surface, their diffusion and dilution pose a challenge to the sensitivity of sensors. As the cell surface is turned into sensor surface, these molecules could be captured prior to their diffusion. This immediate interaction enhances the sensitivity of secretion detection. Various cell secretions have been studied previously

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

utilizing cell-surface affinity sensors, including ATP [91, 92], growth factors [93], cytokines [94], and antigens [95]. Liu et al. [96] developed a detection method that utilized fluorescent magnetic nanoparticles labeled secondary antibody, and achieved a detection limit of 0.1 pg/mL. The cell surface was biotinylated, and functionalized by neutravidin and a biotinylated IL-6 capture antibody. Upon the binding of cytokines secreted from cells, fluorescent magnetic nanoparticles labeled secondary antibodies are introduced for indication of the amount of cytokine secretion.

A challenge of cell-surface affinity sensors is the damage and cell viability caused by cell surface modification [97]. Due to the high background signal from free labeled secondary antibody, the cells after secretion have to undergo wash steps to remove free secondary antibodies. This limits its application on the real-time monitoring of cell secretion.
