Journal of Chromatography B, 1021 (2016) 14–29 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom epage: www.elsevier.com/locate/chromb Review An emerging micro-scale immuno-analytical diagnostic tool to see the unseen. Holding promise for precision medicine and P4 medicine Norberto A. Guzman a,b,* , Daniel E. Guzman b,1 a Princeton Biochemicals, Inc., Princeton, NJ 08543, USA b AffinityCE LLC, East Brunswick, NJ 08816, USA a r t i c l e i n f o Article history: Received 25 August 2015 Received in revised form 15 November 2015 Accepted 17 November 2015 Available online 23 November 2015 Keywords: Reviews Bioanalysis Biomarkers Immunoaffinity capillary electrophoresis Point-of-care instrumentation Precision medicine P4 medicine Solid-phase extraction Circulating tumor cells Exosomes a b s t r a c t Over the years, analytical chemistry and immunology have contributed significantly to the field of clinical diagnosis by introducing quantitative techniques that can detect crucial and distinct chemical, bio- chemical and cellular biomarkers present in biosamples. Currently, quantitative two-dimensional hybrid immuno-analytical separation technologies are emerging as powerful tools for the sequential isolation, separation and detection of protein panels, including those with subtle structural changes such as vari- ants, isoforms, peptide fragments, and post-translational modifications. One such technique to perform this challenging task is immunoaffinity capillary electrophoresis (IACE), which combines the use of anti- bodies and/or other affinity ligands as highly selective capture agents with the superior resolving power of capillary electrophoresis. Since affinity ligands can be polyreactive, i.e., binding and capturing more than one molecule, they may generate false positive results when tested under mono-dimensional pro- cedures; one such application is enzyme-linked immunosorbent assay (ELISA). IACE, on the other hand, is a two-dimensional technique that captures (isolation and enrichment), releases, separates and detects (quantification, identification and characterization) a single or a panel of analytes from a sample, when coupled to one or more detectors simultaneously, without the presence of false positive or false negative data. This disruptive technique, capable of preconcentrate on-line results in enhanced sensitivity even in the analysis of complex matrices, may change the traditional system of testing biomarkers to obtain more accurate diagnosis of diseases, ideally before symptoms of a specific disease manifest. In this manuscript, we will present examples of the determination of biomarkers by IACE and the design of a miniaturized multi-dimensional IACE apparatus capable of improved sensitivity, specificity and throughput, with the potential of being used as a point-of-care instrument and holding promise for precision medicine and P4 medicine. © 2015 Elsevier B.V. All rights reserved. Abbreviations: ACM, analyte concentrator-microreactor; AR, auxiliary reser- voir (buffer container); -NGF, beta-nerve growth factor; BR, buffer reservoir (buffer container); BDNF, brain-derived neurotrophic factor; CA, capillary adap- tor (at point of detection); CE, capillary electrophoresis; CSF, cerebrospinal fluid; CNTF, ciliary neurotrophic factor; DC, detection capillary; IACE, immunoaffinity cap- illary electrophoresis; IgG, immunoglobulin G; Fab, fragment antigen binding; Fc, fragment crystallizable; FO, fiber optic; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LIF, laser-induced fluorescence; LH, luteiniz- ing hormone; M, mandril (capillary extender); MS, mass spectrometry; NT-3, neurotrophin-3; NT-4, neurotrophin-4; SC, separation capillary; TC, transport capil- lary; TSH, thyroid-stimulating hormone; UV, ultraviolet; V, micro-valve; WR, waste reservoir (waste container). * Corresponding author at: Princeton Biochemicals, Inc., P.O. Box 7102, Princeton, NJ 08543, USA. E-mail address: guzman@affinityce.com (N.A. Guzman). 1 Daniel Guzman, B.Sc., was an intern at AffinityCE LLC during the writing of this manuscript. 1. Introduction Without a doubt, the most complex machinery and information- processing system in existence is the human body [1,2]. It is a highly engineered and programmed chemical plant providing a coordinated and regulated environment to facilitate the balanced functionality of the entire being [3,4]. This chemical plant is com- posed of trillions of cells, each of which contain a wide range of chemical and biochemical entities [5]. Cells can absorb and secrete chemicals from and into biological fluids, which in part are indi- cators, or biomarkers, of the balanced functionality of the body, predicting wellness, illness, disability and death [6–8]. Most of the time, this sophisticated machine runs just fine on its own; however, things may go wrong and early detection of warning signs can be the key to preventing a total breakdown. As a con- sequence, there has been a rapid growth of scientific endeavors in http://dx.doi.org/10.1016/j.jchromb.2015.11.026 1570-0232/© 2015 Elsevier B.V. All rights reserved.