The integrated on-chip isolation and detection of circulating tumour cells.

Circulating tumour cells (CTCs) are cancer cells shed from a primary tumour which intravasate into the blood stream and have the potential to extravasate into distant tissues, seeding metastatic lesions. As such, they can offer important insight into cancer progression with their presence generally associated with a poor prognosis. The detection and enumeration of CTCs is, therefore, critical to guiding clinical decisions during treatment and providing information on disease state. CTC isolation has been investigated using a plethora of methodologies, of which immunomagnetic capture and microfluidic size-based filtration are the most impactful to date. However, the isolation and detection of CTCs from whole blood comes with many technical barriers, such as those presented by the phenotypic heterogeneity of cell surface markers, with morphological similarity to healthy blood cells, and their low relative abundance (∼1 CTC/1 billion blood cells). At present, the majority of reported methods dissociate CTC isolation from detection, a workflow which undoubtedly contributes to loss from an already sparse population. This review focuses on developments wherein isolation and detection have been integrated into a single-step, microfluidic configuration, reducing CTC loss, increasing throughput, and enabling an on-chip CTC analysis with minimal operator intervention. Particular attention is given to immune-affinity, microfluidic CTC isolation, coupled to optical, physical, and electrochemical CTC detection (quantitative or otherwise).

Alternating Magnetic Field-Promoted Nanoparticle Mixing: The On-Chip Immunocapture of Serum Neuronal Exosomes for Parkinson's Disease Diagnostics.

The analysis of cargo proteins in exosome subpopulations has considerable value in diagnostics but a translatable impact has been limited by lengthy or complex exosome extraction protocols. We describe herein a scalable, fast, and low-cost exosome extraction using an alternating (AC) magnetic field to support the dynamic mixing of antibody-coated magnetic beads (MBs) with serum samples within 3D-printed microfluidic chips. Zwitterionic polymer-coated MBs are, specifically, magnetically agitated and support ultraclean exosome capture efficiencies >70% from <50 µL of neat serum in 30 min. Applied herein to the immunocapture of neuronal exosomes using anti-L1CAM antibodies, prior to the array-based assaying of α-synuclein (α-syn) content by a standard duplex electrochemical sandwich ELISA, sub pg/mL detection was possible with an excellent coefficient of variation and a sample-to-answer time of ∼75 min. The high performance and semiautomation of this approach hold promise in underpinning low-cost Parkinson's disease diagnostics and is of value in exosomal biomarker analyses more generally.

Multiplexed electrochemical assays for clinical applications.

Rapid, accurate diagnoses are central to future efficient healthcare to identify diseases at early stages, avoid unnecessary treatment, and improve outcomes. Electrochemical techniques have been applied in many ways to support clinical applications by enabling the analysis of relevant disease biomarkers in user-friendly, sensitive, low-cost assays. Electrochemistry offers a launchpad for multiplexed biomarker assays that offer more accurate and precise diagnostics compared to single biomarker assays. In this short review, we underpin the importance of multiplexed analyses and provide a universal overview of current electrochemical assay strategies for multiple biomarkers. We highlight relevant examples of electrochemical methods that successfully quantify important disease biomarkers. Finally, we offer a future outlook on possible strategies that can be employed to increase throughput, sensitivity, and specificity of multiplexed electrochemical assays.

Ultrafast Biomarker Quantification through Reagentless Capacitive Kinetics.

We introduce a facile assessment of binding kinetics at bioreceptive redox-active interfaces as a means of quantifying target proteins. This is achieved by monitoring the redox capacitance (Cr) of a receptor-modified conductive polymer interface under continuous flow. Exemplified with the quantification of C-reactive protein (CRP), capacitance analyses resolve both the association and dissociation regimes in real-time. Significantly, the rate of electrochemical signal change within the association regime is a sensitive function of target concentration, enabling marker assaying down to picomolar levels, comparable to end-point assays, in 15 s. This reagentless proof-of-principle methodology is envisioned to be widely applicable to the facile quantification of a range of other pertinent, clinically relevant targets.

Catalysed amplification of faradaic shotgun tagging in ultrasensitive electrochemical immunoassays.

We introduce a novel electrochemical protein quantitation based on the shotgun biotin tagging of proteins prior to their interfacial immunocapture and polymeric enzyme tagging. The highly amplified faradaic signals generated from a novel ferrocene-tyramine adduct enable fg mL-1 (attomolar) levels of detection and span cross a 5 orders of magnitude dynamic range. This work supports ultrasensitive protein marker detection in a single antibody immunoassay format.

Characterising the biosensing interface.

The development of diagnostic devices relies heavily on the immobilization of biomolecules on supportive substrates, that is the generation of interfaces that can thereafter produce a quantifiable signal upon exposure to a specific target. The ability of a biosensor to selectively recruit analytes is highly dependent on the quality of this receptive biolayer and its functionality. Key performance metrics are selectivity and sensitivity and both are highly dependent on the interfacial structural and physical properties, though often these are not well resolved; in many cases analyses are performed, for example with little knowledge of receptor surface coverage, orientation and/or distribution. In this review, we provide a, necessarily concise, but comprehensive summary of accessible and relevant characterization techniques, noting operational principles, limitations, and the value they can bring in optimising downstream sensor performance.

A Quantification of Target Protein Biomarkers in Complex Media by Faradaic Shotgun Tagging.

The progressive emergence of protein biomarkers promises a revolution in the healthcare industry and a shift of focus from disease management to much earlier intervention. Here, we introduce a facile shotgun tagging of ensemble proteins in clinically relevant media prior to specific target capture at antibody-modified electrodes. This facilitates a convenient voltammetric quantification of markers down to sub-pg/mL levels and across several orders of concentration. A translation of the methodology to an automated microfluidic platform enables marker quantification from 25 µL of sample in less than 15 min, demonstrated here with a simultaneous assaying of CRP and cardiac troponin I (cTnI). The assays show a good correlation with a standard immunoassay when applied to real patient serum samples. The platform is simple, generic, highly sensitive and requires no secondary labeling/binding or amplification.

Real-time Voltammetric Anion Sensing Under Flow.

The development of real-life applicable ion sensors, in particular those capable of repeat use and long-term monitoring, remains a formidable challenge. Herein, we demonstrate, in a proof-of-concept, the real-time voltammetric sensing of anions under continuous flow in a 3D-printed microfluidic system. Electro-active anion receptive halogen bonding (XB) and hydrogen bonding (HB) ferrocene-isophthalamide-(iodo)triazole films were employed as exemplary sensory interfaces. Upon exposure to anions, the cathodic perturbations of the ferrocene redox-transducer are monitored by repeat square-wave voltammetry (SWV) cycling and peak fitting of the voltammograms by a custom-written MATLAB script. This enables the facile and automated data processing of thousands of SW scans and is associated with an over one order-of-magnitude improvement in limits of detection. In addition, this improved analysis enables tuning of the measurement parameters such that high temporal resolution can be achieved. More generally, this new flow methodology is extendable to a variety of other analytes, including cations, and presents an important step towards translation of voltammetric ion sensors from laboratory to real-world applications.

Open Circuit Potential as a Tool for the Assessment of Binding Kinetics and Reagentless Protein Quantitation.

A microfluidic open circuit potential label-free protein assay was developed for the reagentless quantification of C-reactive protein (CRP), a model protein target, and further utilized to assess target-receptor binding kinetics. Generated sensors have very high baseline stabilities (<1% change in 100 min) and high levels of selectivity in complex media. Real-time assays are fast (<20 min), of high sensitivity (1 ng/mL limit of detection for CRP in serum), and resolve kinetic and thermodynamic characteristics that correlate well with those resolved optically. The assay shows excellent correlation with an enzyme-linked immunosorbent assay analysis of patient samples. The methodology has value in potentially underpinning a low-cost, rapid, and sensitive single-step biomarker quantification.

Detecting cancer metastasis and accompanying protein biomarkers at single cell levels using a 3D-printed microfluidic immunoarray.

A low-cost microfluidic microarray capable of lysing cells and quantifying proteins released after lysis was designed and 3D-printed. The array lyses cells on-chip in lysis buffer augmented with a 2s pulse of a sonic cell disruptor. Detection of desmoglein 3 (DSG3), a metastatic biomarker for head and neck squamous cell carcinoma (HNSCC), along with two accompanying HNSCC biomarkers from a single cell lysate of oral cancer cell cultures was demonstrated. A lysis chamber and reagent compartments deliver sample and reagents into detection chambers decorated with capture antibodies immobilized onto inner walls coated with a highly swollen 3D chitosan hydrogel film. Sandwich immunoassays are achieved when captured analytes labeled with biotinylated secondary antibodies, which then capture streptavidin-poly [horse radish peroxidase] (Poly-HRP). Subsequent delivery of super-bright femto-luminol with H2O2 generates chemiluminescence captured with a CCD camera. DSG3 is membrane-bound protein in HNSCC cells of invaded lymph nodes, vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-C (VEGF-C) were positive controls overexpressed into the HNSCC culture medium. Beta-tubulin (ß-Tub) was used as a loading control to estimate the number of cells in analyzed samples. Limits of detection (LOD) were 0.10 fg/mL for DSG3, and 0.20 fg/mL for VEGF-A, VEGF-C and ß-Tub. Three orders of magnitude semilogarithmic dynamic ranges were achieved. VEGF-A showed high in-cell expression, but VEGF-C had low levels inside cells. The very low LODs enabled quantifying these proteins released from single cells. Strong correlation between results from on-chip cell lysis, conventional off-line lysis and ELISA confirmed accuracy.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 µL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

3D-Printed Immunosensor Arrays for Cancer Diagnostics.

Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers' and clinicians' needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as additive manufacturing, has drastically reduced the time of sensor and sensor array development while offering excellent sensitivity at a fraction of the cost of conventional technologies such as photolithography. In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.

Perioperative Outcomes of Open Retrograde Extraperitoneal Versus Intracorporeal Robot-assisted Radical Cystoprostatectomy in Men: A Dual-center Comparative Study.

INTRODUCTION: We compared retrograde extraperitoneal open radical cystoprostatectomy (REORC) and robot-assisted radical cystoprostatectomy with intracorporeal diversion (iRARC) and have reported the early perioperative outcomes. PATIENTS AND METHODS: REORC and iRARC were each performed at a different tertiary high-volume center in 2 countries. Men aged ≥ 18 years with precystectomy clinical stage T1-T3 disease were included. Patients with previous major pelvic and/or intra-abdominal surgery, those who had undergone previous pelvic and/or abdominal irradiation, women, and patients with clinical stage T4 disease were excluded. All cases were managed according to a standardized enhanced recovery after surgery protocol, and all the patients had undergone ileal conduit urinary diversion. Bowel recovery was one of the main endpoints; thus, the intervals to passing flatus, tolerating oral feeding, and bowel opening were determined. The operative time, estimated blood loss, intraoperative complications, length of hospital stay, postcystectomy tumor type, stage, margin status, lymph node yield, and 30- and 90-day complications were analyzed. RESULTS: We performed a retrospective analysis of prospectively collected data from October 2016 to December 2018 of 99 patients, 50 of whom had undergone REORC and 49 iRARC. The demographic data and preoperative parameters were comparable between the 2 groups. REORC resulted in a significantly shorter mean operative time (P < .001), significantly greater mean estimated blood loss (P < .001), and greater percentage of patients requiring blood transfusion (98% vs. 12.24%). No significant differences in the length of stay were observed (P = .412). The rate of prolonged postoperative ileus was 16% and 18.4% in the REORC and iRARC groups, respectively (P = .3). Differences in the interval to passing flatus, tolerating solid oral intake, and bowel opening were not statistically significant between the 2 groups (P = .423, P = .770, and P = .700, respectively). No statistically significant difference was observed in the postcystectomy pathologic outcomes and overall and major complications rates at 30 and 90 days. CONCLUSION: REORC resulted in quicker bowel recovery and a shorter length of stay compared with conventional open procedures, with advantages comparable to those realized with iRARC. Thus, REORC can be adopted as the preferred open approach at institutions without surgical robots available.

Bifocal Compression-Distraction for Combined Bone and Soft-Tissue Defects in Post-traumatic Tibial Nonunion.

OBJECTIVE: To compare 2 distraction osteogenesis techniques in post-traumatic tibial nonunion patients with composite bone and soft-tissue defects. DESIGN: Nonrandomized prospective, case series, single-center study. SETTING: Department of Orthopaedics and Traumatology, Limb Reconstruction Unit, El-Helal hospital, Cairo, Egypt. PARTICIPANTS: Fifty post-traumatic tibial nonunion patients with composite bone and soft-tissue defects. INTERVENTION: Twenty-five patients were treated using bone transport (BT) technique, and 25 patients were treated using acute shortening (AS) and distraction technique. OUTCOME MEASUREMENTS: The external fixation index (EFI); functional and bone results; and complication rates. RESULTS: All patients were followed for a minimum of 18 months after removal of their Ilizarov frame. AS and BT groups were followed up for a mean of 19.7 and 20.3 months, respectively. The mean bone gap after resection and debridement was 4 cm in AS group and 5.9 cm in BT group (P = 0.06). The mean EFI was statistically significant and lower in the AS group compared with BT group (P = 0.03). There were no other statistically significant differences between either intervention groups. CONCLUSIONS: Both techniques achieved comparable good to excellent results, and the differences in number of complications and ASAMI scores for bone or function were not statistically significant. Yet, it appears that the AS technique may be superior because it has a significantly lower EFI. This may not be feasible in all cases, however, because the AS technique is limited by the defect size and the condition of the surrounding soft tissues. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Influence of antibody immobilization strategy on carbon electrode immunoarrays.

We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL-1, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL-1, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.

Accessible Telemedicine Diagnostics with ELISA in a 3D Printed Pipette Tip.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 µL pipettes. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.

An Ultra-Shapeable, Smart Sensing Platform Based on a Multimodal Ferrofluid-Infused Surface.

The development of wearable, all-in-one sensors that can simultaneously monitor several hazard conditions in a real-time fashion imposes the emergent requirement for a smart and stretchable hazard avoidance sensing platform that is stretchable and skin-like. Multifunctional sensors with these features are problematic and challenging to accomplish. In this context, a multimodal ferrofluid-based triboelectric nanogenerator (FO-TENG), featuring sensing capabilities to a variety of hazard stimulus such as a strong magnetic field, noise level, and falling or drowning is reported. The FO-TENG consists of a deformable elastomer tube filled with a ferrofluid, as a triboelectric layer, surrounded by a patterned copper wire, as an electrode, endowing the FO-TENG with excellent waterproof ability, conformability, and stretchability (up to 300%). In addition, The FO-TENG is highly flexible and sustains structural integrity and detection capability under repetitive deformations, including bending and twisting. This FO-TENG represents a smart multifaceted sensing platform that has a unique capacity in diverse applications including hazard preventive wearables, and remote healthcare monitoring.

3D-Printed Biosensor Arrays for Medical Diagnostics.

While the technology is relatively new, low-cost 3D printing has impacted many aspects of human life. 3D printers are being used as manufacturing tools for a wide variety of devices in a spectrum of applications ranging from diagnosis to implants to external prostheses. The ease of use, availability of 3D-design software and low cost has made 3D printing an accessible manufacturing and fabrication tool in many bioanalytical research laboratories. 3D printers can print materials with varying density, optical character, strength and chemical properties that provide the user with a vast array of strategic options. In this review, we focus on applications in biomedical diagnostics and how this revolutionary technique is facilitating the development of low-cost, sensitive, and often geometrically complex tools. 3D printing in the fabrication of microfluidics, supporting equipment, and optical and electronic components of diagnostic devices is presented. Emerging diagnostics systems using 3D bioprinting as a tool to incorporate living cells or biomaterials into 3D printing is also reviewed.

Automated 4-Sample Protein Immunoassays using 3D-Printed Microfluidics.

Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.00 using 1-2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL-1 for PSA and 230 fg mL-1 for PSMA, with dynamic ranges up to 5 ng mL-1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab's website.