Fluorometric determination of ciprofloxacin using molecularly imprinted polymer and polystyrene microparticles doped with europium(III)(DBM)3phen.

The authors describe a microparticle-based system for the detection of the fluoroquinolone antibiotic ciprofloxacin. The method is using the tris(dibenzoylmethane)(1,10-phenanthroline)europium(III) luminophore in polystyrene microparticles along with a molecularly imprinted polymer (MIP) for ciprofloxacin. If ciprofloxacin is captured by the MIP, it quenches the fluorescence of the luminophores. Fluorescence drops linearly in the 0.5-100 µg L-1 ciprofloxacin concentration range, and the detection limit is 92 ng L-1. The method was applied to the analysis of fish samples to assess the analytical performance of the probe. Recoveries ranged from 85.4 to 86.6%, and relative standard deviations between 2.1 and 3.9% (for n = 5). Graphical abstract Schematic presentation of a microparticle-based probe using the tris(dibenzoylmethane)(1,10-phenanthroline)europium(III) luminophore in polystyrene particles along with a molecularly imprinted polymer for ciprofloxacin. After removal of template, carboxylic groups left in the probe can bind to ciprofloxacin through hydrogen bonds.

Hybrid Paper-Plastic Microchip for Flexible and High-Performance Point-of-Care Diagnostics.

A low-cost and easy-to-fabricate microchip remains a key challenge for the development of true point-of-care (POC) diagnostics. Cellulose paper and plastic are thin, light, flexible, and abundant raw materials, which make them excellent substrates for mass production of POC devices. Herein, a hybrid paper-plastic microchip (PPMC) is developed, which can be used for both single and multiplexed detection of different targets, providing flexibility in the design and fabrication of the microchip. The developed PPMC with printed electronics is evaluated for sensitive and reliable detection of a broad range of targets, such as liver and colon cancer protein biomarkers, intact Zika virus, and human papillomavirus nucleic acid amplicons. The presented approach allows a highly specific detection of the tested targets with detection limits as low as 102 ng mL-1 for protein biomarkers, 103 particle per milliliter for virus particles, and 102 copies per microliter for a target nucleic acid. This approach can potentially be considered for the development of inexpensive and stable POC microchip diagnostics and is suitable for the detection of a wide range of microbial infections and cancer biomarkers.

DNA engineered micromotors powered by metal nanoparticles for motion based cellphone diagnostics.

HIV-1 infection is a major health threat in both developed and developing countries. The integration of mobile health approaches and bioengineered catalytic motors can allow the development of sensitive and portable technologies for HIV-1 management. Here, we report a platform that integrates cellphone-based optical sensing, loop-mediated isothermal DNA amplification and micromotor motion for molecular detection of HIV-1. The presence of HIV-1 RNA in a sample results in the formation of large-sized amplicons that reduce the motion of motors. The change in the motors motion can be accurately measured using a cellphone system as the biomarker for target nucleic acid detection. The presented platform allows the qualitative detection of HIV-1 (n = 54) with 99.1% specificity and 94.6% sensitivity at a clinically relevant threshold value of 1000 virus particles/ml. The cellphone system has the potential to enable the development of rapid and low-cost diagnostics for viruses and other infectious diseases.

Nanoparticle-enhanced electrical detection of Zika virus on paper microchips.

Zika virus (ZIKV) is a reemerging flavivirus causing an ongoing pandemic and public health emergency worldwide. There are currently no effective vaccines or specific therapy for Zika infection. Rapid, low-cost diagnostics for mass screening and early detection are of paramount importance in timely management of the infection at the point-of-care (POC). The current Zika diagnostics are laboratory-based and cannot be implemented at the POC particularly in resource-limited settings. Here, we develop a nanoparticle-enhanced viral lysate electrical sensing assay for Zika virus detection on paper microchips with printed electrodes. The virus is isolated from biological samples using antibodies and labeled with platinum nanoparticles (PtNPs) to enhance the electrical signal. The captured ZIKV-PtNP complexes are lysed using a detergent to release the electrically charged molecules associated with the intact virus and the PtNPs on the captured viruses. The released charged molecules and PtNPs change the electrical conductivity of the solution, which can be measured on a cellulose paper microchip with screen-printed microelectrodes. The results confirmed a highly specific detection of ZIKV in the presence of other non-targeted viruses, including closely related flaviviruses such as dengue virus-1 and dengue virus-2 with a detection limit down to 101 virus particles per µl. The developed assay is simple, rapid, and cost-effective and has the potential for POC diagnosis of viral infections and treatment monitoring.

Motion-Based Immunological Detection of Zika Virus Using Pt-Nanomotors and a Cellphone.

Zika virus (ZIKV) infection is an emerging pandemic threat to humans that can be fatal in newborns. Advances in digital health systems and nanoparticles can facilitate the development of sensitive and portable detection technologies for timely management of emerging viral infections. Here we report a nanomotor-based bead-motion cellphone (NBC) system for the immunological detection of ZIKV. The presence of virus in a testing sample results in the accumulation of platinum (Pt)-nanomotors on the surface of beads, causing their motion in H2O2 solution. Then the virus concentration is detected in correlation with the change in beads motion. The developed NBC system was capable of detecting ZIKV in samples with virus concentrations as low as 1 particle/µL. The NBC system allowed a highly specific detection of ZIKV in the presence of the closely related dengue virus and other neurotropic viruses, such as herpes simplex virus type 1 and human cytomegalovirus. The NBC platform technology has the potential to be used in the development of point-of-care diagnostics for pathogen detection and disease management in developed and developing countries.

Ultrasensitive DNA Detection with Hydrodynamic Separation of Plasmonic Nanoparticles and Isothermal Amplification.

Nucleic acid based assays are routinely used to detect diseases and monitor medical treatment. Here, we demonstrated a novel approach for colorimetric DNA detection using plasmonic gold nanoparticles (AuNPs) as hydrodynamic separators coupled with differential centrifugation. This approach relies upon the change in the sedimentation rate of AuNPs when conjugated to DNA amplicons. Isothermal nucleic acid amplification results in the formation of unique DNA amplicons that is large enough to prevent the sedimentation of conjugated AuNPs at a specific centrifugal force. In contrast, free nanoparticles are readily centrifuged and the solution color changes to colorless, enabling accurate and quantitative detection of the targeted DNA. This approach was challenged for the detection of sdfI gene of Salmonella. The decline of the red color intensity of AuNPs was linear to the concentration of the targeted DNA from 1.2 × 101 copies/ml to 1.2 × 107 copies/ml and the detection limit was as low as 120 copies/ml (S/N = 3). This simple platform could be used to establish inexpensive and sensitive assays for clinical and in-field diagnostic applications.

Applications of gold nanoparticles in virus detection.

Viruses are the smallest known microbes, yet they cause the most significant losses in human health. Most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. Therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. The introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. Gold nanoparticles (AuNPs) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. Here, we review the applications of AuNPs in virus testing and detection. The developed AuNP-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-AuNP hybrid structures, virus detection targets, and assay modalities and formats. We pay particular attention to highlighting the functional role and activity of each core Au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. In addition, we provide a general summary of the contributions of AuNPs to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications.

Electrically Oscillating Plasmonic Nanoparticles for Enhanced DNA Vaccination against Hepatitis C Virus.

The promise of DNA vaccines is far-reaching. However, the development of potent immunization methods remains a key challenge for its use in clinical applications. Here, an approach for in vivo DNA vaccination by electrically activated plasmonic Au nanoparticles is reported. The electrical excitation of plasmonic nanoparticles can drive vibrational and dipole-like oscillations that are able to disrupt nearby cell membranes. In combination with their intrinsic ability to focus and magnify the electric field on the surface of cells, Au nanoparticles allow enhanced cell poration and facilitate the uptake of DNA vaccine. Mice immunized with this approach showed up to 100-fold higher gene expression compared to control treatments (without nanoparticles) and exhibited significantly increased levels of both antibody and cellular immune responses against a model hepatitis C virus DNA vaccine. This approach can be tuned to establish controlled and targeted delivery of different types of therapeutic molecules into cells and live animals as well.

Rapid, label-free CD4 testing using a smartphone compatible device.

The most recent guidelines have called for a significant shift towards viral load testing for HIV/AIDS management in developing countries; however point-of-care (POC) CD4 testing still remains an important component of disease staging in multiple developing countries. Advancements in micro/nanotechnologies and consumer electronics have paved the way for mobile healthcare technologies and the development of POC smartphone-based diagnostic assays for disease detection and treatment monitoring. Here, we report a simple, rapid (30 minutes) smartphone-based microfluidic chip for automated CD4 testing using a small volume (30 µL) of whole blood. The smartphone-based device includes an inexpensive (<$5) cell phone accessory and a functionalized disposable microfluidic device. We evaluated the performance of the device using spiked PBS samples and HIV-infected and uninfected whole blood, and compared the microfluidic chip results with the manual analysis and flow cytometry results. Through t-tests, Bland-Altman analyses, and regression tests, we have shown a good agreement between the smartphone-based test and the manual and FACS analysis for CD4 count. The presented technology could have a significant impact on HIV management in developing countries through providing a reliable and inexpensive POC CD4 testing.

An automated smartphone-based diagnostic assay for point-of-care semen analysis.

Male infertility affects up to 12% of the world's male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones.

Paper microchip with a graphene-modified silver nano-composite electrode for electrical sensing of microbial pathogens.

Rapid and sensitive point-of-care diagnostics are of paramount importance for early detection of infectious diseases and timely initiation of treatment. Here, we present cellulose paper and flexible plastic chips with printed graphene-modified silver electrodes as universal point-of-care diagnostic tools for the rapid and sensitive detection of microbial pathogens or nucleic acids through utilizing electrical sensing modality and loop-mediated isothermal amplification (LAMP). We evaluated the ability of the developed paper-based assay to detect (i) viruses on cellulose-based paper microchips without implementing amplification in samples with viral loads between 106 and 108 copies per ml, and (ii) amplified HIV-1 nucleic acids in samples with viral loads between 10 fg µl-1 and 108 fg µl-1. The target HIV-1 nucleic acid was amplified using the RT-LAMP technique and detected through the electrical sensing of LAMP amplicons for a broad range of RNA concentrations between 10 fg µl-1 and 108 fg µl-1 after 40 min of amplification time. Our assay may be used for antiretroviral therapy monitoring where it meets the sensitivity requirement of the World Health Organization guidelines. Such a paper microchip assay without the amplification step may also be considered as a simple and inexpensive approach for acute HIV detection where maximum viral replication occurs.

Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis.

As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants.

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay.

Convenient and rapid immunofiltration assays (IFAs) enable on-site "yes" or "no" determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.

The Quality of In Vivo Upconversion Fluorescence Signals Inside Different Anatomic Structures.

Fluorescence imaging is a broadly interesting and rapidly growing strategy for non-invasive clinical applications. However, because of interference from light scattering, absorbance, and tissue autofluorescence, the images can exhibit low sensitivity and poor quality. Upconversion fluorescence imaging, which is based on the use of near-infrared (NIR) light for excitation, has recently been introduced as an improved approach to minimize the effects of light scattering and tissue autofluorescence. This strategy is promising for ultrasensitive and deep tissue imaging applications. However, the emitted upconversion fluorescence signals are primarily in the visible range and are likely to be absorbed and scattered by tissues. Therefore, different anatomic structures could impose various effects on the quality of the images. In this study, we used upconversion-core/silica-shell nanoprobes to evaluate the quality of upconversion fluorescence at different anatomic locations in athymic nude mice. The nanoprobe contained an upconversion core, which was green (ß-NaYF4:Yb3+/Ho3+) or red (ß-NaYF4:Yb3+/Er3+), and a nonporous silica shell to allow for multicolor imaging. High-quality upconversion fluorescence signals were detected with signal-to-noise ratios of up to 170 at tissue depths of up to - 1.0 cm when a 980 nm laser excitation source and a bandpass emission filter were used. The presence of dense tissue structures along the imaging path reduced the signal intensity and imaging quality, and nanoprobes with longer-wavelength emission spectra were therefore preferable. This study offers a detailed analysis of the quality of upconversion signals in vivo inside different anatomic structures. Such information could be essential for the analysis of upconversion fluorescence images in any in vivo biodiagnostic and microbial tracking applications.

Nanoparticle-mediated systemic delivery of siRNA for treatment of cancers and viral infections.

RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short interfering RNA (siRNA) therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and silicon-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Challenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review.

Hybrid nanocluster plasmonic resonator for immunological detection of hepatitis B virus.

Approximately 88% of the world population lives in regions with intermediate to high incidence of Hepatitis B virus (HBV), yet current serological and DNA-based detection methods have limited sensitivity and convenience. Here, we describe a preassembled plasmonic resonance nanocluster for HBV detection. The gold nanoparticle acceptors (AuNPs), with HBV surface antigen (HBsAg) epitope, and quantum dot (QD) donors with Fab antibody, are assembled into an immuno-mediated 3D-oriented complex with enhanced energy transfer and fluorescence quenching. The coherent plasmonic resonance between Au and QD nanoparticles is exploited to achieve improved donor-acceptor resonance within the nanocluster, which in the presence of HBV viral particles is disassembled in a highly specific manner. The nanocluster provides high detection specificity and sensitivity of HBV, with a sensitivity limit down to 1-100 viral particles per microliter and to attomolar levels of HBsAg. This general platform could be used to establish multiplex diagnostic assays for a variety of other microbial pathogens.