A multiplexed immunochemical microarray for the determination of cardiovascular disease biomarkers

Abstract

A fluorescence antibody microarray has been developed for the determination of relevant cardiovascular disease biomarkers for the analysis of human plasma samples. Recording characteristic protein molecular fingerprints to assess individual's states of health could allow diagnosis to go beyond the simple identification of the disease, providing information on its stage or prognosis. Precisely, cardiovascular diseases (CVDs) are complex disorders which involve different degenerative processes encompassing a collection of biomarkers related to disease progression or stage. The novel approach that we propose is a fluorescent microarray chip has been developed accomplishing simultaneous determination of the most significant cardiac biomarkers in plasma aiming to determine the CVD status stage of the patient. As proof of concept, we have chosen five relevant biomarkers, C-reactive protein (CRP) as biomarker of inflammation, cystatin C (CysC) as biomarker of renal failure that is directly related with heart failure, cardiac troponin I (cTnI) as already established biomarker for cardiac damage, heart fatty acid binding protein as biomarker of ischemia (H-FABP), and finally, NT-proBNP (N-terminal pro-brain natriuretic peptide), a well-established heart failure biomarker. After the optimization of the multiplexed microarray, the assay allowed the simultaneous determination of 5 biomarkers in a buffer solution reaching LODs of 15 ± 5, 3 ± 1, 24 ± 3, 25 ± 3, and 3 ± 1 ng mL^-1, for CRP, CysC, H-FABP, cTnI, and NT-proBNP, respectively. After solving the matrix effect, and demonstrating the accuracy for each biomarker, the chip was able to determine 24 samples per microarray chip. Then, the microarray has been used on a small pilot clinical study with 29 plasma samples from clinical patients which suffered different CVD and other related disorders. Results show the superior capability of the chip to provide clinical information related to the disease in terms of turnaround time (1 h 30 min total assay and measurement) and amount of information delivered in respect to reference technologies used in hospital laboratories (clinical analyzers). Despite the failure to detect c-TnI at the reported threshold, the microarray technology could be a powerful approach to diagnose the cardiovascular disease at early stage, monitor its progress, and eventually providing information about an eminent potential risk of suffering a myocardial infarction. The microarray chip here reported could be the starting point for achieving powerful multiplexed diagnostic technologies for the diagnosis of CVDs or any other pathology for which biomarkers have been identified at different stages of the disease.

Keywords: Acute myocardial infarction; Fluorescence detection; Heart failure; Immunoassay; Inflammation fluorescence; Microarray; Multiplexation.

Fig. 1

The entire pathophysiology of acute coronary syndrome (ACS). This flowchart depicts candidate markers related to earlier aspects of atherogenesis which may provide independent information in the diagnosis of AMI. CRP C-reactive protein, IL-6 interleukin-6, TNFα tumor necrosis factor α, MMP-2/-9 matrix metalloproteinases 2 and 9, MPO myeloperoxidase, ICAM intracellular cell adhesion molecules, VCAM vascular cell adhesion molecules, PAPP-A pregnancy-associated plasma protein A, IMA ischemia-modified albumin, uFFA unbound free fatty acids, H-FABP heart-type-isoform fatty acid binding protein, sCD40L soluble CD40 ligand, PlGF placental growth factor, TnT troponin T, cTnI cardiac troponin I, CK-MB creatine kinase MB isoenzyme, BNP brain natriuretic peptide, NT-proBNP N-terminal pro-brain natriuretic peptide. Figure adapted from [6]

Fig. 2

Evaluation of the cTnI and cTnI in I-T-C complex non-specific adsorption when 0.15% casein was present in the assay buffer (PBST). Both cTnI forms were assayed at 0.15 µg mL⁻¹, using a [Abcapt] = 0.5 mg/mL and As220 as Abdet at 1/1000. The standard deviation shown is the result of analysis made 1 day using 6 well replicates

Fig. 3

Calibration curves for each analyte: CRP, Cys C, H-FABP, cTnITC complex, and NT-proBNP in PBST 0.15% casein pH 7.5 in a multiplexed configuration

Fig. 4

Correlation between the fortified and measured concentration values for A CRP, B Cys C, C H-FABP, D Tn I-T-C complex, and E NT-proBNP assays. CRP and Cys C are performed in one slide with 1/40 sample dilution, while H-FABP, troponin, and NT-proBNP are performed in another slide without sample dilution. The dotted line corresponds to a perfect correlation (slope = 1)

Fig. 5

Results from the clinical plasma samples performed in the multiplexed microarray platform. Light gray bars correspond to measurements taken by the already characterized samples by IGTP. H-FABP, cTnI, and NT-proBNP were measured undiluted and CRP and Cys C was measured dilution 1/40 in PBST 0.15% casein pH 7.5. In the right y-axis, it is described the limit of detection (LOD) and working range, the basal concentration, and the established concentration found in the cardiovascular diseases for each biomarker