PHYSICAL REVIEW E 94, 042408 (2016) Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples Logeeshan Velmanickam, Darrin Laudenbach, and Dharmakeerthi Nawarathna Department of Electrical and Computer Engineering, North Dakota State University, Fargo, North Dakota, 58102-6050, USA (Received 15 June 2016; published 11 October 2016) The current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The detection limit of ELISA is about 250 pg/ml. However, to quantify analytes that are related to various stages of tumors including early detection requires detecting well below the current limit of the ELISA test. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are <100 pg/ml and the prostate specific antigen level of the early stage of prostate cancer is about 1 ng/ml. Further, it has been reported that there are significantly less than 1 pg/mL of analytes in the early stage of tumors. Therefore, depending on the tumor type and the stage of the tumors, it is required to quantify various levels of analytes ranging from ng/ml to pg/ml. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes (<pg/ml). To address this gap, we here report on a label-free, high-throughput technique based on dielectrophoresis. This technique is capable of quantifying target analytes down to a few thousands of molecules (∼zmoles). DOI: 10.1103/PhysRevE.94.042408 I. INTRODUCTION Immunoassays are utilized to detect or quantify target biomolecules such as proteins, antigens, and antibodies in biological samples [1,2]. Typically, in immunoassays, first the specific target antibody is attached onto a solid surface of a device or a traditional well plate. Second, the biological sample that is containing the target molecules (analytes) is pipetted or flowed over the antibodies allowing the analytes to conjugate with antibodies. Finally, the presence of antibody-analyte complexes is detected and the levels of target analytes in the sample can be quantified [2,3]. For example, during the diagnosis of tumors or when monitoring the progress of ongo- ing treatments for tumors, it is required to monitor the levels of representative tumor markers (proteins) in patients’ blood [4,5]. This is typically performed through immunoassays, and the current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The ELISA uses antibody-analyte conjugation followed by quantification of antibody-analyte complexes in the sample [5]. The commonly used method for quantifying antibody-analyte complexes involves measuring the concentration (analyte) dependent color change or fluores- cence intensity change in the sample. The detection limit of ELISA is about 250 pg/ml [6]. However, to detect or quantify analytes (proteins) that are related to various stages of tumors, including early detection, requires detecting well below the current limit of the ELISA. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are <100 pg/ml and the prostate specific antigen (PSA) level of the early stage of prostate cancer is about 1 ng/ml [7]. Further, it has been reported that there are ≪pg/mL analytes in the early stage of tumors [7]. Therefore, depending on the tumor type and the stage of the tumors, various levels of analytes ranging from ng/ml to pg/ml must be quantified [6,7]. Furthermore levels of the number of protein targets (typically four to six protein targets) must be detected or quantified in a single experiment [8]. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes (<pg/ml). To address this critical need in biology and medicine, a number of new techniques have been proposed and utilized. Among the new techniques, impedimetric based analyte detection or quantification offers a low cost and label-free technique [8–11]. It uses an array of microelectrodes called interdigitated electrodes that are fabricated on glass or similar materials [12–14]. In impedimetric experiments, the change of impedance upon binding the target analytes onto antibodies that are immobilized on the electrodes or between electrodes is measured at low frequencies (<1000 Hz). Using a standard curve of known analyte concentration vs change in impedance, the unknown analyte concentrations are calculated [13]. It has been reported that the lowest analyte concentration that can detect or quantify using this technique is about 80 pg/ml [8]. However, impedimetric based analyte detection or quantifica- tion suffers a number of limitations, such as the following: Impedance is dependent on the conductivity of the biological sample, there is need for expensive electric circuits and equip- ment (impedance analyzers) to record the impedance, and impedance varies from one analyte type to another. In addition to impedimetric based detection or quantification, there are number of other techniques such as ion sensitive field-effect transistors, semiconducting carbon nanotubes, thin-film gate transistors, and electrolyte-insulator-semiconductor structures that are available to detect or quantify target analytes of a sample [8,15]. However, almost all of these techniques require target analytes in very low ionic buffer solutions. Therefore, these techniques have been used in applications such as detecting DNA molecules and DNA hybridization events in low-conductivity buffers. Furthermore, it has also been reported that successful development of these techniques for DNA analysis is much complex than expected. In particular, the theoretical basis of the observed results, including a wide variety of reported signal amplitudes and response times, still remains unclear [15]. Therefore, these techniques have very limited applicability in immunoassays. To address this gap, we report here on a label-free, high-throughput technique that is capable of detecting or quantifying target analytes down to few thousands of molecules (∼zmoles). Furthermore, our technique can be integrated with microfluidics chips for 2470-0045/2016/94(4)/042408(6) 042408-1 ©2016 American Physical Society