Paper-Based All-in-One Origami Nanobiosensor for Point-of-Care Detection of Cardiac Protein Markers in Whole Blood

Abstract

Rapid and accurate diagnosis of cardiovascular diseases (CVDs) at the earliest stage is of paramount importance to improve the treatment outcomes and avoid irreversible damage to a patient’s cardiovascular system. Microfluidic paper-based devices (μPADs) represent a promising platform for rapid CVD diagnosis at the point of care (POC). This paper presents an electrochemical μPAD (E-μPAD) with an all-in-one origami design for rapid and POC testing of cardiac protein markers in whole blood. Based on the label-free, electrochemical impedance spectroscopy (EIS) immunoassay, the E-μPAD integrates all essential components on a single chip, including three electrochemical cells, a plasma separation membrane, and a buffer absorption pad, enabling easy and streamlined operations for multiplexed detection of three cardiac protein markers [cardiac troponin I (cTnI), brain natriuretic peptide (BNP)-32, and D-Dimer] on a finger-prick whole blood sample within 46 min. Superior analytical performance is achieved through sensitive EIS measurement on carbon electrodes decorated with semiconductor zinc oxide nanowires (ZnO NWs). Using spiked human plasma samples, ultralow limits of detection (LODs) of E-μPAD are achieved at 4.6 pg/mL (190 fM) for cTnI, 1.2 pg/mL (40 fM) for BNP-32, and 146 pg/mL (730 fM) for D-Dimer. Real human blood samples spiked with purified proteins are also tested, and the device’s analytical performance was proven to be comparable to commercial ELISA kits. The all-in-one E-μPAD will allow rapid and sensitive testing of cardiac protein markers through easy operations, which holds great potential for on-site screening of acute CVDs in nonlaboratory settings such as emergency rooms, doctor’s offices, or patient homes.

Advancements in electrochemical biosensing of cardiovascular disease biomarkers

“…Utilizing electrochemical impedance spectroscopy measurements of zinc oxide nanowires, the E-μPAD can sensitively measure key proteins such as cTnI, BNP-32, and D-Dimer, demonstrating its potential for rapid CVD screening outside traditional laboratory settings. 25…”

Extending the Shelf-Life of Immunoassay-Based Microfluidic Chips through Freeze-Drying Sublimation Techniques

“…This necessitates the development of an all-in-one dry chemistry microfluidic chip for robust POCT applications [32]. Protein conformations and antibody functionalities are highly sensitive to their surrounding environments [13,33]. As a result, the surface chemistry in the channels of these devices tends to degrade over time, particularly when exposed to harsh conditions like high temperatures and high humidity, common in tropical regions where they are frequently utilized for POCT [34].…”

“…Freeze-drying, also known as lyophilization or cryo-desiccation, is a preservation method that involves controlled freezing followed by the sublimation of ice under a vacuum. This technique has been traditionally employed across various industries such as pharmaceuticals, food, and biotechnology to preserve temperature-sensitive materials like bovine serum albumin (BSA) [32], food products [33], and antibodies [31]. However, applying freeze-drying methods to microfluidic devices presents unique challenges, particularly concerning the impact on materials used for device fabrication, such as PMMA, glass slides, and double-sided adhesive films.…”