Approaching near real-time biosensing: Microfluidic microsphere based biosensor for real-time analyte detectionby Noa Cohen, Pooja Sabhachandani, Alexander Golberg, Tania Konry

Biosensors and Bioelectronics


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Author’s Accepted Manuscript

Approaching near real-time biosensing:

Microfluidic microsphere based biosensor for realtime analyte detection

Noa Cohen, Pooja Sabhachandani, Alexander

Golberg, Tania Konry

PII: S0956-5663(14)00907-5


Reference: BIOS7284

To appear in: Biosensors and Bioelectronic

Received date: 25 August 2014

Revised date: 28 October 2014

Accepted date: 12 November 2014

Cite this article as: Noa Cohen, Pooja Sabhachandani, Alexander Golberg and

Tania Konry, Approaching near real-time biosensing: Microfluidic microsphere based biosensor for real-time analyte detection, Biosensors and Bioelectronic,

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Approaching near real-time biosensing: Microfluidic microsphere based biosensor for realtime analyte detection

Noa Cohen †**, Pooja Sabhachandani †**, Alexander Golberg ‡, Tania Konry*† † Department of Pharmaceutical Sciences, Northeastern University, 140 The Fenway, Room 441/ 446, 360 Huntington Avenue, Boston, MA, 02115 ‡ Centre for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School,

Shriners Burns Institute, Boston, Massachusetts, United States of America **Equal contribution. * Corresponding author:


In this study we describe a simple lab-on-a-chip (LOC) biosensor approach utilizing well mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics of clinically relevant analytes such cytokines and antibodies. We were able to overcome the adsorption kinetics reaction rate-limiting mechanism, which is diffusion-controlled in standard immunoassays, by introducing the microsphere-based assay into well-mixed yet simple microfluidic device with turbulent flow profiles in the reaction regions. The integrated microsphere-based LOC device performs dynamic detection of the analyte in minimal amount of biological specimen by continuously sampling micro-liter volumes of sample per minute to detect dynamic changes in target analyte concentration. Furthermore we developed a mathematical model for the well-mixed reaction to describe the near real time detection mechanism observed in the developed LOC method. To demonstrate the specificity and sensitivity of the developed real time monitoring LOC approach, we applied the device for clinically relevant analytes: Tumor Necrosis Factor (TNF)-α cytokine and its clinically used inhibitor, anti-TNF-α antibody. Based on the reported results herein, the developed LOC device provides continuous sensitive and specific near real-time monitoring method for analytes such as cytokines and antibodies, reduces reagent volumes by nearly three orders of magnitude as well as eliminates the washing steps required by standard immunoassays.

Keywords : Microsphere, real time detection, lab on a chip, TNF-α, cytokine , antibody 1. Introduction

Rapid, sensitive and quantitative detection methods of disease markers are necessary for timely and effective diagnosis and therapy (Martinez et al., 2008). A major challenge in the detection of soluble molecules such as cytokines, protein antigens and antibodies is the ability to monitor time-varying or dynamic concentrations in real-time. Currently there are no online monitoring approaches available for continuous analyte immunoassays and pharmacokinetic characterization of biomolecules in real-time. At present, state-of-the-art analyte detection techniques include immunoassays such as enzyme-linked immunosorbent assays (ELISA) and Luminex assays, which are based on specific recognition of clinical antigens by the respective antibodies (Reichert, 2001, Djoba Siawaya et. al., 2008). These diagnostic methods are performed on samples obtained at pre-defined times and are therefore laborious and time-intensive procedures.

Additionally, these methods are impractical for real-time monitoring since they cannot be performed rapidly enough to assess dynamic fluctuation of analyte concentration in vivo. This limits their utility in clinical settings where it is of critical importance to generate real-time profile of analytes such as cytokines or administered drugs in vivo (Crowther, 2001; Mannerstedt et al., 2010; Mao et al., 2009; Wild, 2001).

In non-mixed solutions, in immunosorbent assays like ELISA, the binding reaction rates for reagents with low binding equilibrium constant, such as high affinity antibody-antigen interaction, depend on diffusion (Porstmann et al., 1992). Further increase of reaction surface or decrease of reaction volumes will not decrease the reaction time (Crowther, 2001). Therefore most, if not all, non-mixing immunoassay systems require incubation of 1-2 hours for analyte detection (Kusnezow et al., 2006; Ruslinga et al., 2010). Several developments in microfluidic based immunosorbent assay have been reported within the past ten years to address the circumventing problems associated with conventional immunoassays (Chen et al., 2011; Hou and Herr, 2010; Lee et al. 2009; Ng et al., 2010; Ng et al. 2012; Nie et al., 2014; Rissin et al, 2010; Thaitrong et al, 2013; Bange et al.). In the microfluidic immunoassay format increased surface area to volume ratios speeds up the antibody–antigen reactions while the smaller dimensions reduce the consumption of expensive reagents and precious samples (Kai et al., 2012; Thaitrong et al, 2013). Never the less most of these methods still require incubation and are unable to measure dynamic changes in the analyte concentration in real time (Hu and Gao, 2007; Singhal et al., 2010).