Cancer-derived extracellular vesicle (EV) nanoparticles carry important biomarkers but are difficult to recover from plasma, making EV-based diagnostics a challenge for clinical settings. Here, we demonstrate nanoparticle-based detection of pancreatic cancer using dielectrophoresis (DEP) nanoparticle recovery technology, which purifies nanoparticles from undiluted plasma and quantifies associated biomarkers. We combined both nanoparticle recovery and biomarker quantification on a single device by simultaneously collecting cell-free DNA nanoparticles and EVs followed by on-chip biomarker fluorescent staining for DNA and Glypican-1. Using a blinded cohort, these biomarkers differentiated pancreatic cancer from benign pancreatic diseases, including cysts, pancreatitis, and precancerous low-grade intraductal papillary mucinous neoplasm (IPMN) lesions, with a sensitivity of 0.92, a specificity of 0.83, and an AUC of 0.93. The AUC increased to 0.97 for patients over 50 years old. This is higher than the standard invasive endoscopic ultrasound-guided fine needle aspiration tissue biopsy procedure (AUC 0.79). This study is among the first demonstrating a combined threshold of DNA and protein levels that can distinguish pancreatic cancer from its precursor IPMN lesions. We also demonstrated the detection of early-stage pancreatic cancer and high-grade in situ precancerous lesions. This DEP-based technique shows that multiple types of cancer-derived nanoparticles can be quickly and easily recovered from plasma making it promising for future clinical diagnostics.

Circulating cell free DNA (cfDNA) is a valuable source of biomarkers for a range of medical applications including detection and monitoring of diseases. Currently, cfDNA sequence analysis must take place in a laboratory setting, due to the multiple steps required for processing including collection, purification, amplification, and analysis. Developing a point-of-care test system that combines these steps would simplify DNA processing thereby increasing diagnostic screening accessibility and enabling real-time monitoring for individual patients. Here, we have developed a system that combines multiple cfDNA processing steps into a single microfluidicbased device. This includes cfDNA collection directly from undiluted human plasma followed by purification and on chip amplification. A microelectrode array embedded within the microfluidic chip collected cfDNA through the creation of dielectrophoretic (DEP) forces followed by a wash to achieve purification. DEP utilizes differences in dielectric properties between cfDNA and plasma to preferentially induce a force on cfDNA. We then achieved on-chip amplification of collected DNA by designing a thermal cycling system to enable polymerase chain reaction (PCR) directly on the chip. This successfully consolidated the most labor-intensive steps of collection, purification, and amplification into a single device. Compared to elution of cfDNA for off-chip amplification, our on-chip PCR method improved the lower limit of detection by 3-fold and improved the total DNA yield by 5-fold. Furthermore, we demonstrate its clinical diagnostic potential by detecting KRAS mutations from a pancreatic ductal adenocarcinoma patient using only 60 mu L of plasma. This paves the way for future development of a fully self-contained system facilitating the rapid detection of mutations in cfDNA.

Protease activity is an emerging biomarker for cancer detection as activity levels are often increased in tumor tissue compared to healthy tissue. Of particular interest is the activity of proteases carried by extracellular vesicle (EV) nanoparticles which are oversecreted by tumors into circulation. Current methods to analyze the activity of proteases bound to EVs require complex multi-instrument sample processing to separate EVs from plasma to quantify protease activity. This makes EV-based protease activity detection a challenge for diagnostic or point-of-care applications. Here, a method is reported that manipulates EV nanoparticles and charged molecular byproducts from protease activity using two different electrokinetic phenomena generated by a single electrode microarray within a microfluidic channel. Dielectrophoresis is first generated to recover EVs carrying active trypsin-like proteases from human plasma followed by electrophoresis for subsequent analysis of peptide cleavage products indicating protease activity. This method demonstrates signal amplification through protease catalytic activity in combination with concentrating mechanisms of dielectrophoresis and electrophoresis. Using this approach, a significant difference in protease activity is observed between patients with pancreatic cancer and benign cysts. This demonstrates dual-electrokinetic chip-based technology as a useful tool to manipulate different sized and charged analytes in a single device enabling future clinical translation of EV-based protease diagnostics.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies.

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low‐volume liquid biopsy assay called cyc‐DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on‐chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on‐chip staining with fluorescently conjugated primary antibodies. cyc‐DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof‐of‐concept technique. We then used cyc‐DEP to quantify blood plasma levels of target proteins from healthy individuals, low‐grade and high‐grade prostate cancer patients ( n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low‐volume liquid biopsies.

The 20th century has seen tremendous innovation of dielectrophoresis (DEP) technologies, with applications being developed in areas ranging from industrial processing to micro- and nanoscale biotechnology. From 2010 to present day, there have been 981 publications about DEP. Of over 2600 DEP patents held by the United States Patent and Trademark Office, 106 were filed in 2019 alone. This review focuses on DEP-based technologies and application developments between 2010 and 2020, with an aim to highlight the progress and to identify potential areas for future research. A major trend over the last 10 years has been the use of DEP techniques for biological and clinical applications. It has been used in various forms on a diverse array of biologically derived molecules and particles to manipulate and study them including proteins, exosomes, bacteria, yeast, stem cells, cancer cells, and blood cells. DEP has also been used to manipulate nano- and micron-sized particles in order to fabricate different structures. The next 10 years are likely to see the increase in DEP-related patent applications begin to result in a greater level of technology commercialization. Also during this time, innovations in DEP technology will likely be leveraged to continue the existing trend to further biological and medical-focused applications as well as applications in microfabrication. As a tool leveraged by engineering and imaginative scientific design, DEP offers unique capabilities to manipulate small particles in precise ways that can help solve problems and enable scientific inquiry that cannot be addressed using conventional methods.

Background: Technology platforms that afford biomarker discovery in patients suffering from traumatic brain injury (TBI) remain an unmet medical need. Here, we describe an observational pilot study to explore the utility of an alternating current electrokinetic (ACE) microchip device in this context. Methods: Blood samples were collected from participating subjects with and without minor TBI. Plasma levels of glial fibrillary acidic protein (GFAP), Tau, ubiquitin C-terminal hydrolase L1 (UCH-L1), and cell-free DNA (cfDNA) were determined in subjects with and without minor TBI using ACE microchip device followed by on-chip immunofluorescent analysis. Post-concussive symptoms were assessed using the Rivermead Post Concussion Symptoms Questionnaire (RPCSQ) at one-month follow-up. Results: Highest levels of GFAP, UCH-L1, and Tau were seen in two minor TBI subjects with abnormality on head computed tomography (CT). In patients without abnormal head CT, Tau and GFAP levels discriminated between plasma from minor-TBI and non-TBI patients, with sensitivity and specificity of 64-72 and 50%, respectively. Plasma GFAP, UCH-L1, and Tau strongly correlated with the cumulative RPCSQ score. Plasma UCH-L1 and GFAP exhibited highest correlation to sensitivity to noise and light (r = 0.96 and 0.91, respectively, p < 0.001). Plasma UCH-L1 and Tau showed highest correlation with headache (r = 0.74 and 0.78, respectively, p < 0.001), sleep disturbance (r = 0.69 and 0.84, respectively, p < 0.001), and cognitive symptoms, including forgetfulness (r = 0.76 and 0.74, respectively, p < 0.001), poor concentration (r = 0.68 and 0.76, respectively, p < 0.001), and time required for information processing (r = 0.77 and 0.81, respectively, p < 0.001). cfDNA exhibited a strong correlation with depression (r = 0.79, p < 0.01) and dizziness (r = 0.69, p < 0.01). While cfDNA demonstrated positive correlation with dizziness and depression (r = 0.69 and 0.79, respectively, p < 0.001), no significant correlation was observed between cumulative RPCSQ and cfDNA (r = 0.07, p = 0.81). Conclusion: We provide proof-of-principle results supporting the utility of ACE microchip for plasma biomarker analysis in patients with minor TBI.

For a programmable mask-less patterning, we develop the novel electrophoretic deposition (EPD) method by a macroscale programmable patterning system, which has a large matrix EPD array device (2.5 × 2.5 mm2 electrodes with 10 μm gap; 20 by 20 electrodes pattern) and printed circuit board controllers. We demonstrate programmed diverse micro/nano size (20 nm – 10 μm) polystyrene beads pattern onto our 400 sites wafer scale chip, which carry out that individually penetrate electrical fields at each electrode. To pattern diverse materials onto non-conductive other substrate, 200 nm nano-porous nitrocellulose membrane is laid onto the system. Cy3 17 bases DNA and 40 nm biotin-coated beads with fluorophore are patterned onto nitrocellulose substrate. Furthermore, we demonstrate that 7–15 nm diameters and 0.5–10 μm lengths carbon nanotubes (CNT) are patterned onto the nitrocellulose substrate by the system. The CNT pattern films are simply lift off from the system. Also, CNT, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) and (6,6)-phenyl-C61-butyric acide methyl ester and poly(3-hexylthiophene) are locally patterned, through the multi-layering process by the system. The multi-layered polymer device is assembled with indium tin oxide onto polyethylene terephthalate as photodetector. The polymer photodetector is demonstrated by light on (2.5 × 10−5 A at 3 V)/off condition. (3.5 × 10−5 A at 3 V). Finally, we demonstrate the fabrication of energy storage device, which is patterned LiCoO2 (LCO)/CNT mixtures/CNT and graphite/CNT as a cathode and anode of secondary battery. The half-cell of 143.7 μm deposited LCO mixtures is successfully operated with 112 mA/g capacity for 3 times charging/discharging cycles.

Though the advances in microelectronic device fabrication have realized new capabilities in integrated analytical and diagnostic platforms, there are still notable limitations in point-of-care sample preparation. AC electrokinetic devices, especially those leveraging dielectrophoresis (DEP), have shown potential to solve these limitations and allow for sample-to-answer in a single point-of-care device. However, when working directly with whole blood or other high conductance (~ 1 S/m) biological fluids, the aggressive electrochemical conditions created by the electrode can fundamentally limit the device operation. In this study, platinum wire-based electrode devices spanning circular polytetrafluorethylene (PTFE) wells and a planar microarray device with sputtered platinum electrodes were tested in plasma and PBS buffers of differing concentration across a wide range of frequencies and electric field intensities (AC voltages) to determine their respective safe regions of operation and to gain an understanding about the failure mechanisms of this class of device. At frequencies of 10 kHz and below, the upper bound of operation is the degradation of electrodes due to electrochemical attack by chlorine overcoming the native platinum oxide passivation. At higher frequencies, 100 kHz and above, the dielectric loss and subsequent heating of the buffer will boil before the electrodes suffer observable damage, due to the slow irreversible reaction kinetics. Effective dielectrophoretic capture of small biological particles at these frequencies is limited, and heat/oxidative denaturation of target material are a major concern. A new class of smaller devices, ones capable of high throughput at voltages low enough to maintain the integrity of the platinum passivation layer, is needed to mitigate these fundamental limitations.

Tumor derived extracellular vesicles, including exosomes, carry cancer related proteins on their outer surfaces making them a valuable source of tumor biomarkers in blood. However, these vesicles are difficult to recover from plasma and this prevents them from being widely used for clinical diagnostic tests. Here we present a technique that uses high conductance dielectrophoresis to rapidly recover these vesicles from plasma allowing for immunofluorescence detection of cancer related biomarkers. We demonstrate this technique can be used to detect biomarkers that successfully distinguish patients with pancreatic cancer from those with benign pancreatic disease, such as pancreatitis, as well as healthy individuals. What makes dielectrophoresis different from other vesicle recovery techniques is that it takes advantage of the large contrast in the dielectric properties between the vesicles and the surrounding plasma. The dielectrophoretic force preferentially draws vesicles to an electrode array at the bottom of a microfluidic chip where they are held in place allowing a fluidic wash to remove the bulk plasma. The vesicles are concentrated at the electrode edge thereby increasing the signal to noise ratio of the fluorescent immunostaining signal and placing the particles in known locations allowing for automated detection and quantification of biomarker levels. This technique takes 30 min to complete, requires 30-50 µl of plasma, and can be highly automated to reduce labor effort making it a promising technology for future translation into the clinical laboratory setting and enabling the use of extracellular vesicles and exosomes for diagnostic applications. Citation Format: Augusta Modestino, Jesus Bueno Alvarez, Michael Heller, Stuart Ibsen. Detection of pancreatic cancer using rapid dielectrophoresis based recovery of tumor derived extra cellular vesicles and exosomes from plasma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4603.

Objective: To examine resting and postprandial peripheral protease activity in healthy controls and individuals with type 2 diabetes mellitus (T2DM) and pre-T2DM. Methods: Individuals with T2DM or pre-T2DM and healthy controls (mean age 55.8 years) were studied before and for a span of 300 minutes following a single high-calorie McDonald's breakfast. Metalloproteases-2/-9 (MMP-2/-9), elastase, and trypsin activities were assessed in whole blood before and following the meal using a novel high-precision electrophoretic platform. Also assessed were circulating levels of inflammatory biomarkers and insulin receptor density on peripheral blood mononuclear cells (PBMCs) in relationship to protease activity. Results: Premeal MMP-2/-9 and elastase activity levels in T2DM and in pre-T2DM participants were significantly elevated as compared to controls. The T2DM group showed a significant increase in elastase activity 15 minutes after the meal; elastase activity continued to increase to the 30-minute time point (p < 0.01). In control participants, MMP-2/-9, elastase, and trypsin were significantly increased at 15 minutes after the meal (p < 0.05) and returned to premeal values within a period of approximately 30 to 60 minutes post meal. PBMCs incubated for 1 hour with plasma from T2DM and pre-T2DM participants had significantly lower levels of insulin receptor density compared to those incubated with plasma from control participants (p < 0.001). Conclusions: The results of this study suggest that individuals with T2DM and pre-T2DM have higher resting systemic protease activity than nonsymptomatic controls. A single high-calorie/high-carbohydrate meal results in further elevations of protease activity in the systemic circulation of T2DM and pre-T2DM, as well as in healthy controls. The protease activity in turn can lead to a downregulation of insulin receptor density, potentially supporting a state of insulin resistance.

Extracellular vesicles (EVs) are small, membrane-bound particles released by all cells that have emerged as an attractive biomarker platform. We study the utility of a dielectrophoretic (DEP) micro-chip device for isolation and characterization of EVs derived from plasma specimens from patients with brain tumors. EVs were isolated by DEP chip and subjected to on-chip immunofluorescence (IF) staining to determine the concentration of glial fibrillary acidic protein (GFAP) and Tau. EVs were analyzed from the plasma samples isolated from independent patient cohorts. Glioblastoma cell lines secrete EVs enriched for GFAP and Tau. These EVs can be efficiently isolated using the DEP platform. Application of DEP to clinical plasma samples afforded discrimination of plasma derived from brain tumor patients relative to those derived from patients without history of brain cancer. Sixty-five percent (11/17) of brain tumor patients showed higher EV-GFAP than the maximum observed in controls. Ninety-four percent (16/17) of tumor patients showed higher EV-Tau than the maximum observed in controls. These discrimination thresholds were applied to plasma isolated from a second, independent cohort of 15 glioblastoma patients and 8 controls. For EV-GFAP, we observed 93% sensitivity, 38% specificity, 74% PPV, 75% NPV, and AUC of 0.65; for EV-Tau, we found 67% sensitivity, 75% specificity 83% PPV, 55% NPV, and AUC of 0.71 for glioblastoma diagnosis. This proof-of-principle study provides support for DEP-IF of plasma EVs for diagnosis of glioblastoma.

Pancreatic ductal adenocarcinoma (PDAC) typically has nonspecific symptoms and is often found too late to treat. Because diagnosis of PDAC involves complex, invasive, and expensive procedures, screening populations at increased risk will depend on developing rapid, sensitive, specific, and cost-effective tests. Exosomes, which are nanoscale vesicles shed into blood from tumors, have come into focus as valuable entities for noninvasive liquid biopsy diagnostics. However, rapid capture and analysis of exosomes with their protein and other biomarkers have proven difficult. Here, we present a simple method integrating capture and analysis of exosomes and other extracellular vesicles directly from whole blood, plasma, or serum onto an AC electrokinetic microarray chip. In this process, no pretreatment or dilution of sample is required, nor is it necessary to use capture antibodies or other affinity techniques. Subsequent on-chip immunofluorescence analysis permits specific identification and quantification of target biomarkers within as little as 30 min total time. In this initial validation study, the biomarkers glypican-1 and CD63 were found to reflect the presence of PDAC and thus were used to develop a bivariate model for detecting PDAC. Twenty PDAC patient samples could be distinguished from 11 healthy subjects with 99% sensitivity and 82% specificity. In a smaller group of colon cancer patient samples, elevated glypican-1 was observed for metastatic but not for nonmetastatic disease. The speed and simplicity of ACE exosome capture and on-chip biomarker detection, combined with the ability to use whole blood, will enable seamless "sample-to-answer" liquid biopsy screening and improve early stage cancer diagnostics.

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations.

We introduce a facile fabrication process for ultrathin nitrogen-doped graphene quantum dot (NGQD) layers on TiO2 nanoparticles in order to develop high-performance photocatalysts. NGQDs are grown either by graphitization of precursors or direct deposition of premade NGQDs. Photocatalytic performance is shown to be dependent on the thickness of NGQD layers and doping levels of C and N atoms on TiO2 surfaces. The nanocomposites shown to absorb a broad range of visible light and narrow the bandgap by 0.38 eV, resulting in the increased density of photoinduced eletron-hole pairs and efficient charge separation at the interface between the NGQDs and TiO2.

Early detection of brain cancer remains a challenging proposition. We applied a novel alternating current electrokinetic (ACE) chip device that relies on the dielectrophoretic (DEP) force to isolate and analyze the proteomic content of extracellular vesicles (EV) derived from undiluted patient plasma to identify biomarkers for early detection of brain tumors. EVs derived from cultured cells or plasma samples were isolated using the ACE chip. The concentration of intra-vesicular glial fibrillary acidic protein (GFAP) and Tau was determined using immunofluorescence staining. Improvement in model prognostication was quantified using net reclassification improvement (NRI>0) and integrated discrimination improvement (IDI). EVs secreted by cultured brain tumor cells (brain metastasis, meningioma, and glioma) harbored high levels of GFAP and Tau. We isolated EVs from plasma collected from brain tumor patients (5 meningiomas, 5 metastases, 6 gliomas) and 17 non-cancer controls. Compared to controls, plasma EVs from brain tumor patients exhibited greater fluorescence for GFAP (1.94 ± 0.139 vs. 1.28 ± 0.042, p<0.0001) and Tau (4.92 ± 0.43 vs. 1.79 ± 0.11, p<0.0001). Immunofluorescence did not differ between tumor types. Elevated EV GFAP was associated with a sensitivity of 88%, a specificity of 92%, and a AUC of 0.931 (95% CI 0.84–1.022) for brain tumor detection. Similarly, elevated EV Tau was associated with sensitivity of 94%, specificity of 94%, and AUC of 0.948 (95% CI 0.846–1.05) for brain tumor detection. The combination of EV GFAP and Tau improved test discrimination relative to GFAP alone (NRI>0 1.66, 95% CI 1.19–2.13, p<0.001; IDI 0.26, 95% CI 0.11–0.41, p<0.001) or Tau alone (NRI>0 1.54, 95% CI 1.03–2.05, p<0.001; IDI 0.18 0.038-0.32, p=0.013). We have provided proof-of-principle studies to demonstrated the utility of a novel DEP-based technology for minimally invasive brain cancer detection using undiluted patient plasma.