Exosomes are cell-derived nanoscale (30-150 nm) vesicles found in body fluids. It has been reported that exosomes carry cell-of-origin specific nucleic acids, proteins and lipid molecules. Therefore, the potential of exosomes as biomarkers for disease detection has been intensively studied. Before any clinical usage of exosomes as biomarkers, in-depth characterization of exosomes, especially, analysis of inclusion molecules is essential. The majority of recent studies have focused on analyzing exosomal membrane proteins. In this study, we have utilized impedance spectroscopy (EIS) to analyze exosomes, which provides valuable information about the dielectric properties of exosomes. EIS is a label-free technique of characterizing samples suspended in a buffer solution. This study examined the magnitude and phase spectra of the exosomes produced by cultured non-cancerous (hTERT-HPNE) and cancerous (MIA PaCa-2) exosomes. EIS was measured using a microscale electrode device consisting of an FR4, a fiberglass laminate material for printed circuit boards, substrate with 10 ounces of copper, with 35 μm cladding. In addition, circuit models with constant phase element (CPE) for the exosome samples with electrodes were developed and analyzed to support our experimental findings. The results indicate that the impedance phase spectra can, in higher concentration samples, characterize samples using the magnitude and phase of the impedance.
Cancer, obesity, and opioid abuse pose a combined threat to the well-being of the people in the United States, affecting over 70% and costing more than $250 billion per year in medical expenses. The unavailability of sensing technologies addressing the fundamental molecular changes related to disease initiation, progression, and therapeutic interventions is a critical roadblock for successfully combating these diseases/disorders. Recent clinical studies have shown that microRNA (miRNA) in circulating blood could use as a potential biomarker for combating these diseases/disorders because miRNA expression take place first in the biochemical cascade and therefore, miRNA could provide reliable and clinically important information that is superior and appear earlier than other biomarkers. Despite this progress, miRNAs have not yet been translated or utilized in the clinical diagnosis of any disease. This lack of progress is partially due to the differences among and limitations of various detection technologies, which produce inconsistent and inaccurate results. To address this issue, we have developed a low-cost and disposable device that can detect and quantify target miRNA levels. MiRNA detection is an integrated two-step process that used external electric fields to selectively concentrate fluorophore-labeled target miRNAs in nanoscale metallic hotspots within the device and enhance the fluorescence intensity via multiple metal-fluorophore interactions. This paper demonstrates how external electric fields could modulate the radiative decay rate of fluorophore molecules and subsequently enhance the fluorescence intensity.
Biomarker detection and quantification in body fluids is widely utilized in medical diagnosis as it provides useful information for developing treatment strategies. Once attached to target biomarker molecules, fluorescence can be used to detect and quantify the target biomarkers. However, expression of target biomarkers in body fluids is generally weak, and therefore direct utilization of fluorescence for detection with high sensitivity and selectivity is challenging. To address this issue, fluorescence enhancement of target biomarkers has been investigated by several research groups. These studies utilized light-metal-fluorophore interactions and have reported about few thousand-fold fluorescence enhancement. Fluorescence enhancement of few thousand folds enables the detection of molarities up to about nanomolar. However, medical diagnosis, especially early diagnosis, requires detection limit of attomolar. To extend the capabilities of fluorescence enhancement to be applicable in the full range of medical diagnosis, we have used low frequency electric fields (<20 MHz) to further enhance the light-metal-fluorophore interactions. This paper presents the results from our simulation work performed to show how electric fields could modulate the fluorescence enhancement. Moreover, we have found that external electric fields can be used to place the fluorophore molecules outside the quenching regions, align fluorophore dipoles with plasmonic axis of metal and place the fluorophore in the high electric field region of the scattered light. These capabilities could lead to fluorescence enhancement up to about billion-fold that enables attomolar detection. In addition to enhancing the fluorescence, we have also studied the effect of electric fields on localized surface plasmon resonance (LSPR).
We propose the use of negative dielectrophoresis (DEP) spectroscopy as a technique to improve the detection limit of rare analytes in biological samples. We observe a significant dependence of the negative DEP force on functionalized polystyrene beads at the edges of interdigitated electrodes with respect to the frequency of the electric field. We measured this velocity of repulsion for 0% and 0.8% conjugation of avidin with biotin functionalized polystyrene beads with our automated software through real-time image processing that monitors the Rayleigh scattering from the beads. A significant difference in the velocity of the beads was observed in the presence of as little as 80 molecules of avidin per biotin functionalized bead. This technology can be applied in the detection and quantification of rare analytes that can be useful in the diagnosis and the treatment of diseases, such as cancer and myocardial infarction, with the use of polystyrene beads functionalized with antibodies for the target biomarkers.
Dielectrophoresis (DEP) is a commonly used technique in biomedical engineering to manipulate biomolecules. DEP is defined as the force acting on dielectric particles when they are exposed to non-uniform electric fields. DEP effect can be divided in three categories: positive (dielectric particles are attracted to the electrodes), negative, and zero force DEP. The cross-over frequency is the frequency in which the DEP force is equal to zero. The cross-over frequency depends on the conductivity and the permittivity of the particles and of the suspended medium. The DEP cross-over frequency has been utilized in detecting/quantifying biomolecules. A manual procedure is commonly used to estimate the cross-over frequency of biomolecules. Therefore, the accuracy of this detection method is significantly limited. To address this issue, we designed and tested an automated procedure to carry out DEP spectroscopy in dielectric particles dissolved in a biological buffer solution. Our method efficiently measures the effect of the DEP force through a live video feed from the microscope camera and performs real-time image processing. It records the change in the fluorescence emission as the system automatically scans the electric frequency of the function generator over a specified time interval. We demonstrated the effectiveness of the method by extracting the crossover frequencies and the DEP spectrum of polystyrene beads with blue color dye (1000 nm diameter) and green fluorescent polystyrene beads with 500 nm diameter using this procedure. This approach can lead to the development of a biosensor with significantly higher sensitivity than existing detection methods.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.