Label-free cytokine micro- and nano-biosensing towards personalized medicine of systemic inflammatory disorders

Abstract

Systemic inflammatory disorders resulting from infection, trauma, surgery, and severe disease conditions pose serious threats to human health leading to organ dysfunction, organ failure, and mortality. The highly complex and dynamic nature of the immune system experiencing acute inflammation makes immunomodulatory therapy blocking pro-inflammatory cytokines very challenging. Successful therapy requires the ability to determine appropriate anti-cytokine drugs to be delivered at a right dose in a timely manner. Label-free micro- and nano-biosensors hold the potential to overcome the current challenges, enabling cytokine-targeted treatments to be tailored according to the immune status of an individual host with their unique cytokine biomarker detection capabilities. This review studies the recent progress in label-free cytokine biosensors, summarizes their performances and potential merits, and discusses future directions for their advancements to meet challenges towards personalized anti-cytokine drug delivery.

Keywords: Anti-Cytokine Drug Delivery; Field Effect Transistor Nanowire; Label-free Biosensors; Microcantilever; Microfluidics; Optical Resonator; Personalized Medicine; Pro-inflammatory Cytokine; Surface Plasmon Resonance.

Fig. 1

Concept of personalized immunomodulatory therapy for systemic inflammatory disease enabled by rapid cytokine-based immune status monitoring. This concept is analogous to feedback-loop system control theory used in system engineering that controls the behavior of a dynamical system with an input.

Fig. 2

Regime map showing sensitivity versus assay time for different types of label-free biosensors. The term of “sensitivity” refers to limit of detection (LOD) in pg/mL. The top panels show the schematics of the sensor types. The color of each panel frame and of each circled area of data points in the regime map identifies the same corresponding sensor type shown above the panel. The black dashed lines show the theoretical limits of sensitivity and assay time based on antibody-analyte binding kinetics. (left dashed line: lower bounds for LOD and assay time in the analyte binding reaction-limited regime at Da<1; right dashed line: lower bounds for LOD and assay time in the analyte diffusion-limited regime at Da>10.) The light blue region shows the biosensor performance needed for acute inflammatory cytokine secretion measurement to serve timely personalized anti-cytokine drug delivery. FET nanowire, EIS, and LSPR biosensors exhibit sensitivity and assay time approaching the desirable levels. Note that the FET nanowire biosensors in ref. [52] and [53] (marked with stars * in the figure) show exceptional sensitivity and assay time beyond the theoretical limit. The underlying mechanisms are unclear. An analyte mass of 15 kDa was used to convert the sensitivity unit from grams per milliliter to molar concentration. The assay time was determined assuming k_on = 10⁻⁶ M⁻¹·sec⁻¹and k_off = 10⁻³ sec⁻¹, respectively. The top panel figures are reproduced from [86] (FET nanowire), [87] (microcantilever), [88] (ring resonator), [89] (impedance) and [85] (LSPR) with permission.

Fig. 3

Calculated molecular limit of detection as a function of detectable binding ratio for label-free biosensors based on Langmuir isotherm. The detectable binding ratio is defined by the total available binding sites (B_n) divided by the minimum numbers of detectable bound molecular (N_Iod). The grey region shows the LOD of the label-free biosensors estimated based on the values of dissociate constant (K_D) from commonly used cytokine antibodies in literature [, –95].

Fig. 4

Microfluidic integration of label-free biosensors enabling on-chip upstream sample preparation and multiplexed cytokine detection. a. Processes of selectively purifying target biomarker proteins from a whole blood sample using a microfluidic device with capture antibodies immobilized to the device via photocleavable linkers. Reproduced from [35] with permission. Upper left: The valve (red arrow) is open to the waste channel of the empty device before sample loading. Upper right: The antibodies immobilized by the photocleavable linkers selectively capture biomarker proteins from the loaded sample, which is followed by a washing process. Lower left: Ultraviolet irradiation releases the captured proteins with the valve closed. Lower right: The valve is open to the downstream chamber with label-free electrochemical biosensor arrays, and the proteins are detected. b. Multiplexed cytokine detection using silicon microring resonator arrays integrated in a microfluidic system. Reproduced from [38] with permission. c. Multiplexed cytokine detection on a LSPR biosensor-arrayed microfluidic chip. Reproduced from [85] with permission. The microfluidic integration of label-free biosensors in b and c enables massively parallel analysis of multiple cytokine analytes in a rapid manner.

Fig. 5

Integrated microfluidics enabling on-chip cell isolation, enrichment and confinement to achieve cell-based cytokine secretion assay. a. Schematic of a microfluidic immunophenotying assay (MIPA) device with a PDMS micromembrane to achieve on-chip isolation and enrichment of THP-1 cells. Reproduced from [103] with permission. b. A microfluidic platform to achieve label-free cytokine secretion assay using an integrated LSPR biosensor. Endotoxin-stimulated target white blood cells are conjugated by microbeads and mechanically trapped by micropillar arrays of the device for their cytokine secretion analysis. Reproduced from [104] with permission. c. A microfluidic device with a PDMS valve structure actuated by a vacuum pump for cell incubation and electrochemical detection of cell-secreted cytokines in an enclosed microenvironment. Reproduced from [105] with permission. The time-variation amperometric signal plot for IFN-γ detection shows that the microfluidic device can continuously monitor cellular cytokine secretion dynamics. Reproduced from [101] with permission. d. A similar microfluidic device with upstream and downstream microchambers is used to study cytokine-mediated cellular communications. Reproduced from [100] with permission.