ReviewAn emerging micro-scale immuno-analytical diagnostic tool to see the unseen. Holding promise for precision medicine and P4 medicine
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 composed 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 indicators, or biomarkers, of the balanced functionality of the body, predicting wellness, illness, disability and death [6], [7], [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 consequence, there has been a rapid growth of scientific endeavors in the last few years searching for early biomarkers of disease and toxicological conditions. Crucial information is dependent upon accurate quantification, identification and characterization of all circulating endogenous and exogenous chemical and biological substances [9], [10], cells [11], subcellular particles [12], cell-derived or extracellular vesicles [13], [14] and their molecular content [15] in biosamples, present at a wide range of cell subpopulations [16] or concentrations, including those substances found at sub-picomolar concentrations [17], [18].
However, given the complexity of most biological samples, sample preparation has been, and continues to be, one of the critical challenges in bioanalysis. Numerous techniques have been developed over the years to quantify small molecules and biomolecules [9], [10], [19], [20], [21]. Demands for small volumes, shorter running times, reliability, robustness, selectivity, precision, versatility, and sensitivity have been most challenging [22], [23]. Biomarker discovery practically includes tools and technologies that aid in the understanding of prediction, cause, diagnosis, regression and outcome of disease [7]. Particularly in the last decade, there have been enormous efforts to transform conventional biological analysis methods into more efficient and sensitive assays employing miniaturized analytical instruments coupled to powerful detectors, including laser-induced fluorescence and mass spectrometers [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]. A considerable amount of literature has been published in the area of solid-phase extraction and its different versions, specifically their applications in the development of selective and sensitive bioanalytical methods [34], [35], [36], [37], [38], [39], [40]. Immunoaffinity enrichment methods [41], [42], [43], [44], [45], [46], [47], [48], [49], [50] have been developed, primarily as off-line procedures, to capture and purify selected analytes from simple and complex matrices prior to their quantification and identification using analytical separation instruments coupled to one or more detection systems. Immunoaffinity capture methods have also been used to isolate cellular structures, including circulating tumor cells [51], [52] and cell-derived vesicles such as exosomes [53], [54].
The current gold standard in bioanalysis for the quantification of a wide range of protein biomarkers is enzyme-linked immunosorbent assay (ELISA), a mono-dimensional technique that has played a central role in diagnostic and clinical research applications for the last two decades [55], [56], [57], [58], [59], [60]. However, as molecular biology advances and as scientists are able to access new technologies with higher recognition accuracy, classical ELISA methods suffer limitations in analysis time, sample size, equipment cost, and in some cases have emerged with a limit of inaccuracy leading to imprecise biomarker detection and resulting in incorrect diagnosis and treatment. Contradictory results using ELISA have been demonstrated for the determination of the miokine irisin, the pancreatic ductal adenocarcinoma biomarker CUZD1, Leishmania antigens, and the appearance of a cross-reacting human chorionic gonadotropin (hCG)-like antigen in septic shock [58], [61], [62], [63], [64], [65]. Also, false-positive results for urine hCG have been described for membranoproliferative glomerulonephritis, chronic renal failure, adenomyosis, tubo-ovarian abscess, and multiple myeloma [65]. Similarly, using an ELISA test, a false-positive result was found for human immunodeficiency virus (HIV) for a patient with systemic lupus erythematosus [66]. For other examples see Table 1 [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78]. The main reason by which false-positive results are obtained using mono-dimensional immunoassays is because many antibody molecules are polyreactive, meaning that they may recognize structurally unrelated targets in addition to their antigens or target molecules [79], [80], [81], [82]. A resolution for inaccurate results is the use of some hybrid techniques, which are best represented by merging on-line immunoassays and analytical separation instruments to form a single two-dimensional capture and separation system [49], [58], [83], [84], [85], [86], [87], [88], [89], [90]. These two-dimensional immuno-separation systems are rapidly gaining acceptance in bioanalytical applications [37], [91], [92], [93], [94], [95].
One such emerging two-dimensional technology is immunoaffinity capillary electrophoresis (IACE), which is promising to be a solution to the inaccurate results produced by mono-dimensional techniques. IACE is carried out in conventional capillary electrophoresis and in microchip capillary electrophoresis formats [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140]. In contrast with bulky immunoaffinity enrichment-designed instruments or devices that are commonly interfaced with liquid chromatography instruments, for use preferentially in research environments, IACE can be manufactured as a miniaturized immuno-analytical separation instrument capable of being used in clinical environments as a point-of-care instrument with improved sensitivity, specificity and throughput [105], [118], [119], [129], [141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151]. The high resolving power of capillary electrophoresis also permits the identification of minor structural differences in proteins that are very difficult to discern with liquid chromatography analysis. As a result, even if a polyclonal antibody used for enrichment of one particular protein cross-reacts with two or more closely-related structural epitopes of similar proteins, IACE will separate each holomeric form, including protein variants and isoforms, as well as peptide fragments and proteins containing one or more post-translational modifications. Therefore, this unique characteristic of IACE facilitates the generation of accurate and reliable results and diminishes significantly the possibility of obtaining false-positive information because structurally unrelated targets can be identified and differentiated from the target molecules primarily by differences in migration behavior, characteristic absorption peaks, and mass spectrometry profile. Furthermore, precision medicine [152] and P4 medicine [153] are paving the way for something that has the promise to change medicine, and IACE technology may become a powerful and affordable platform for making precision medicine and P4 medicine an achievable reality in the years to come.
Section snippets
General principle of IACE
Since the introduction of IACE in the early 1990s by our laboratory, Terry Phillips’ laboratory and others [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [154], [155], [156], [157], [158], a growing number of scientists have developed many IACE applications, employing conventional CE or microchip CE format, as indicated in Fig. 1.
Emphasis should be made that the term “immunoaffinity capillary electrophoresis” is a broad concept used for the separation of substances
Miniaturized IACE instrument
Advancements in the miniaturization of a myriad of components and matrices necessary for the manufacture of diagnostic instruments has been dependent upon the evolution of micro- and nano-technologies that permit small, light-weight, low-power, cost-efficient, and high-sensitivity instruments to be brought to a patient, or to function in any other place away from a large centralized laboratory, such as a doctor’s office, ambulance, or in the field [223], [224], [225], [226], [227], [228], [229]
Clinical applications of IACE
Examples of clinical applications that utilize the technology of IACE are discussed in this section. It is noteworthy to note that when referring to the process of introducing samples and buffers into a capillary column, the procedure is carried out from the inlet end of the capillary to the outlet end as indicated in Fig. 2. This unidirectional traditional format of sample introduction into a capillary or column is performed for all analytical separation systems. Microchip capillary
Future directions of IACE
The progression from a healthy to a disease state is marked by significant biological changes within an individual. Clinically, presenting symptoms can be non-specific and variable enough to hinder diagnosis, and may appear only after a disease has already become well-established and consequently more difficult to treat [284]. Biomarkers are extremely valuable tools for healthcare professionals looking to overcome such challenges. Changes in biomarker levels can predict clinical outcomes which
Conclusion
Faster, efficient, robust, portable, capable of high-throughput, multi-dimensional, ability to multi-task, easy to operate, and cost effective are some of the main characteristics of a micro-scale immuno-separation instrument. In addition, this type of instrument has the potential to yield accurate, sensitive, and specific biomarkers, and to impact clinical diagnosis. There is now a paradigm shift moving away from traditional diagnostic tests and curative medicine, to predictive, personalized
Disclosure
Dr. Norberto Guzman is an employee of Princeton Biochemicals, Inc. and AffinityCE LLC. He is also the inventor of several patents issued and pending related to the IACE technology. There was no external financial support necessary to conduct the research projects.
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Daniel Guzman, B.Sc., was an intern at AffinityCE LLC during the writing of this manuscript.