Sensitive and simultaneous detection of cardiac markers in human serum using surface acoustic wave immunosensor.

We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on sensing area of the SAW sensor, which could specifically capture and detect cardiac markers (cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibodies. Introduction of these complexes to the capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, a gold staining method was performed, which demonstrated that it is possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. The sensor response due to gold staining varied as a function of cardiac marker concentration. We also investigated effects of increasing operating frequency on sensor responses. Results showed that detection limit of the SAW sensor could be further improved by increasing the operating frequency.

Electrochemical cell lysis device for DNA extraction.

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC) applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH(-)) at the cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device design, were two important parameters of the device performance. After optimizing the design and visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR) analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid processing. The device could potentially be applied as an on-chip DNA extraction component.

Cytosolic delivery mediated via electrostatic surface binding of protein, virus, or siRNA cargos to pH-responsive core-shell gel particles.

We recently described a strategy for intracellular delivery of macromolecules, utilizing pH-responsive "core-shell" structured gel particles. These cross-linked hydrogel particles disrupt endosomes with low toxicity by virtue of physical sequestration of an endosome-disrupting "proton sponge" core inside a nontoxic hydrophilic shell. Here we tested the efficacy of this system for cytosolic delivery of a broad range of macromolecular cargos, and demonstrate the delivery of proteins, whole viral particles, or siRNA oligonucleotides into the cytosol of dendritic cells and epithelial cells via core-shell particles. We assessed the functional impact of particle delivery for vaccine applications and found that cytosolic delivery of protein antigens in dendritic cells via the core-shell particles promotes priming of CD8(+) T-cells at 100-fold lower doses than soluble protein. Functional gene knockdown following delivery of siRNA using the particles was demonstrated in epithelial cells. Based on these findings, these materials may be of interest for a broad range of biomedical applications.

Surface acoustic wave immunosensor for real-time detection of hepatitis B surface antibodies in whole blood samples.

We demonstrate an application of Love wave mode surface acoustic wave (SAW) immunosensor to detect hepatitis B surface antibody (HBsAb) in aqueous conditions. SiO(2) guiding layer was deposited on 36 degrees YX-LiTaO(3) piezoelectric single crystal substrate to protect the electrodes and to trap the acoustic energy near the surface, and hepatitis B surface antigen (HBsAg) was immobilized on the sensing area. The resonance frequency shift was monitored to detect specific binding of HBsAb to immobilized HBsAg. To eliminate the effects of other physical factors except for the mass change, the resonance frequency was compared to that of a reference SAW device coated with bovine serum albumin (BSA) to block binding of HBsAb. The guiding layer thickness with maximum mass sensitivity was found to be 5 microm, which was in agreement with the theoretical calculation, and the center resonance frequency was around 199 MHz. The sensor showed binding specificity to HBsAb and a linear relationship between the frequency shift and the antibody concentration with sensitivity of 0.74 Hz/(pg/microl) and detection limit below 10 pg/microl. In addition, our SAW immunosensor successfully detected HBsAb in whole blood samples without any pretreatment, opening up its applicability in fast label-free protein detection methods.