Comparative analysis of genome code complexity and manufacturability with engineering benchmarks.

When knowledge has advanced to a state that includes a predictive understanding of the relationship between genome sequence and organism phenotype it will be possible for future engineers to design and produce synthetic organisms. However, the possibility of synthetic biology does not necessarily guarantee its feasibility, in much the same way that the possibility of a brute force attack fails to ensure the timely breaking of robust encryption. The size and range of natural genomes, from a few million base pairs for bacteria to over 100 billion base pairs for some plants, suggests it is necessary to evaluate the practical limits of designing genomes of similar complexity. This analysis characterizes the complexity of natural genomes, compares them to existing engineering benchmarks, and shows that existing large software programs are on similar scale with the genome of complex natural organisms.

Electrospinning of cyanoacrylate tissue adhesives for human dural repair in endonasal surgery.

Cerebral spinal fluid (CSF) leakage is a major postoperative complication requiring surgical intervention, resulting in prolonged healing and higher costs. Biocompatible polymers, such as cyanoacrylates, are currently used as tissue adhesives for closing surgical defects and incisions. Coupling these polymers with nanofiber technology shows promising results for generating nanofibers used in wound care, tissue engineering, and drug delivery. Fiber membranes formed by electrospinning of n-octyl-2-cyanoacrylate (NOCA) are investigated for in situ dural closures after neurosurgery to improve the quality of the closure and prevent post-surgical CSF leaks. Electrospun NOCA fiber membranes showed significantly higher sealing capabilities of defects in human dura, with an average burst pressure of 149 mmHg, compared with that of an FDA-approved common dural sealant that had an average burst pressure of 37 mmHg. In this study, microfabrication of NOCA fibers demonstrates a promising technique for dural repairs.

In vitro and in vivo evaluation of microneedles coated with electrosprayed micro/nanoparticles for medical skin treatments.

AIM: Microneedles (MNs) create micropunctures and deliver drugs or nutrients deep into skin layer. We demonstrated that MNs, coated with electrosprayed nanoparticles loaded with functional molecules, are useful for transdermal delivery. METHODS: Electrospraying was utilised to generate drug-loaded nanoparticles and to create uniform coating on MNs. Process parameters and release kinetics were evaluated in vitro. The in vivo efficacy of insulin-coated MNs was investigated using diabetic rats. RESULTS: Electrosprayed micro/nanoparticles loaded with dye or insulin were coated on MNs with particle size of 515 ± 286 and 522 ± 261 nm, respectively. Optimally coated MNs resulted in >70% transfer rate into porcine skins. Insulin-coated MNs were applied to diabetic rats resulting in reduction of blood glucose levels fluctuations, compared to subcutaneous injections. CONCLUSIONS: Electrospraying is shown to be an effective method to coat MNs with drug-loaded nanoparticles. Coated MNs provide a promising platform for cosmetic, drug and protein delivery applications.

Quantitative hematocrit measurement of whole blood in a point-of-care lateral flow device using a smartphone flow tracking app.

We present a rapid and quantitative point-of-care (PoC) system based on a smartphone application that is capable of accurately tracking the flow of red blood cells (RBCs) through a no-reaction lateral flow assay (nrLFA) device. Utilizing only the camera feed from the smartphone and built-in image processing, the nrLFA is identified and RBC fluid flow distances and rates are recorded in parallel with the test without the need of any custom hardware or enclosure. We demonstrated the application by first measuring and then calculating hematocrit (Hct) values of whole blood samples with nominal content of 28%, 35%, 40%, and 45% Hct on the nrLFA platform. The PoC system was able to accurately measure (to within 1% Hct of nominal values) whole blood Hct in ~10-20 s after sample dispensing.

Self-inflating floating nanofiber membranes for controlled drug delivery.

Floating gastro-retentive delivery systems can prolong the gastric residence providing sustained drug release. In this study, we report on self-inflating effervescence-based electrospun nanofiber membranes embedding polyethylene oxide/sodium bicarbonate cast films. In this system, sodium bicarbonate results in an effervescence effect by creating carbon dioxide gas upon contacting an acidic gastric fluid, with the resulting gas bubbles being entrapped within the swollen network of nanofibers. Eudragit RL and RS polymers are utilized as a host material to manipulate release kinetics of incorporated drugs. Pramipexole, a common medication for chronic Parkinson's disease (PD), is used as a model drug. Uniform and bead-free nanofibers with diameters of ~300 nm were obtained. Although floating nanofibers initially exhibited high water contact angles (WCA), water droplets were quickly absorbed into the surface and the WCA decreased to ~0° within 60 s. Floating lag time, total floating time, swelling properties and drug release profiles were investigated both in a simulated gastric fluid (pH 1.2 buffer solution) and in a simulated intestinal fluid (pH 6.8 buffer solution) at 37 °C. All floating nanofiber formulations began to float instantly with nearly zero floating lag time and did not sink into the solution even after 24 h. By comparison, the same formulations without sodium bicarbonate cast films could not maintain continuous floating beyond 15 min. The floating nanofiber pouches presented lower initial release of between 20 and 57 %, compared to that of non-floating nanofiber pouches (40-82% within 2 h). Clearly, floating nanofibers reduced the initial burst release and provided sustained drug release. This demonstrates the potential to result in 'once-a-day' oral introduction of drugs that normally must be taken frequently. Effervescence-based floating nanofibers present a novel and promising prototype delivery system for the drug delivery in the upper gastro-intestinal (GI) tract.

Lateral flow assay using aptamer-based sensing for on-site detection of dopamine in urine.

A lateral flow assay using DNA aptamer-based sensing for the detection of dopamine in urine is reported. Dopamine duplex aptamers (hybridized sensor with capture probe) are conjugated to 40-nm Au nanoparticles (AuNPs) with 20T linkers. The detection method is based on the dissociation of the duplex aptamer in the presence of dopamine, with the sensor part undergoing conformational changes and being released from the capture part. Hybridization between the complementary DNA in the test line and the conjugated AuNP-capture DNA produces a red band, whose intensity is related to the dopamine concentration. The minimum detectable concentration obtained by ImageJ analysis was <10 ng/mL (65.2 nM), while the visual limit of detection is estimated to be ~50 ng/mL (normal range of dopamine in urine of 52-480 ng/mL or 0.3-3.13 µM). No cross reactivity to other stress biomarkers in urine was confirmed. These results indicate that this robust and user-friendly point-of-care biosensor has significant potential for providing a cost-effective alternative for dopamine detection in urine.

Aptamer-Based Lateral Flow Biosensor for Rapid Detection of Salivary Cortisol.

We have developed a disposable point-of-care (POC) aptamer-based biosensor for the detection of salivary cortisol. Nonstressful and noninvasive sampling of saliva compared to that of blood makes saliva an attractive biological matrix in developing POC devices for biomarker monitoring. Aptamers are attractive as recognition elements for multiple reasons, including their specific chemical synthesis, high stability, lack of immunogenicity, and cell-free evolution. A duplex aptamer conjugated to the surface of Au nanoparticles (AuNPs) by Au-S bonds is utilized as the sensor probe in a lateral flow assay (LFA) device. The addition of saliva samples containing cortisol makes the cortisol-aptamer undergo conformational changes and dissociate from the capture probe. Increasing cortisol concentration in the dispensed saliva sample results in increased dissociation and leads to increased binding of AuNP conjugate on the test line. Therefore, the color intensity of the test line on the LFA is a direct function of the concentration of cortisol in saliva. This simple and fast method provides detection in the cortisol range of ∼0.5-15 ng/mL, which is in the clinically accepted range for salivary cortisol. The limit of detection was 0.37 ng/mL, and the accuracy was confirmed by enzyme-linked immunosorbent assay (ELISA) testing results. High selectivity was observed for salivary cortisol against other closely related steroids and stress biomarkers present in saliva.

Multi-layered core-sheath fiber membranes for controlled drug release in the local treatment of brain tumor.

Interstitial chemotherapy plays a pivotal role in the treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain cancer, by enhancing drug biodistribution to the tumor and avoiding systemic toxicities. The use of new polymer structures that extend the release of cytotoxic agents may therefore increase survival and prevent recurrence. A novel core-sheath fiber loaded with the drug carmustine (BCNU) was evaluated in an in vivo brain tumor model. Three-dimensional discs were formed from coaxially electrospun fiber membranes and in vitro BCNU release kinetics were measured. In vivo survival was assessed following implantation of discs made of compressed core-sheath fibers (NanoMesh) either concurrently with or five days after intracranial implantation of 9L gliosarcoma. Co-implantation of NanoMesh and 9L gliosarcoma resulted in statistically significant long-term survival (>150 days). Empty control NanoMesh confirmed the safety of these novel implants. Similarly, Day 5 studies showed significant median, overall, and long-term survival rates, suggesting optimal control of tumor growth, confirmed with histological and immunohistochemical analyses. Local chemotherapy by means of biodegradable NanoMesh implants is a new treatment paradigm for the treatment for brain tumors. Drug delivery with coaxial core-sheath structures benefits from high drug loading, controlled long-term release kinetics, and slow polymer degradation. This represents a promising evolution for the current treatment of GBM.

Label-Free Optical Detection of Multiple Biomarkers in Sweat, Plasma, Urine, and Saliva.

We report a novel label-free quantitative detection of human performance "stress" biomarkers in different body fluids based on optical absorbance of the biomarkers in the ultraviolet (UV) region. Stress biomarker (hormones and neurotransmitters) concentrations in bodily fluids (blood, sweat, urine, saliva) predict the physical and mental state of the individual. The stress biomarkers primarily focused on in this manuscript are cortisol, serotonin, dopamine, norepinephrine, and neuropeptide Y. UV spectroscopy of stress biomarkers performed in the 190-400 nm range has revealed primary and secondary absorption peaks at near-UV wavelengths depending on their molecular structure. UV characterization of individual and multiple biomarkers is reported in various biofluids. A microfluidic/optoelectronic platform for biomarker detection is reported, with a prime focus toward cortisol evaluation. The current limit of detection of cortisol in sweat is ∼200 ng/mL (∼0.5 µM), which is in the normal (healthy) range. Plasma samples containing both serotonin and cortisol resulted in readily detectable absorption peaks at 203 (serotonin) and 247 (cortisol) nm, confirming feasibility of simultaneous detection of multiple biomarkers in biofluid samples. UV spectroscopy performed on various stress biomarkers shows a similar increasing absorption trend with concentration. The detection mechanism is label free, applicable to a variety of biomarker types, and able to detect multiple biomarkers simultaneously in various biofluids. A microfluidic flow cell has been fabricated on a polymer substrate to enable point-of-use/care UV measurement of target biomarkers. The overall sensor combines sample dispensing and fluid transport to the detection location with optical absorption measurements with a UV light emitting diode (LED) and photodiode. The biomarker concentration is indicated as a function of photocurrent generated at the target wavelength.

Coaxial Electrospinning Formation of Complex Polymer Fibers and their Applications.

The formation of fibers by electrospinning has experienced explosive growth in the past decade, recently reaching 4,000 publications and 1,500 patents per year. This impressive growth of interest is due to the ability to form fibers with a variety of materials, which lend themselves to a large and rapidly expanding set of applications. In particular, coaxial electrospinning, which forms fibers with multiple core-sheath layers from different materials in a single step, enables the combination of properties in a single fiber that are not found in nature in a single material. This article is a detailed review of coaxial electrospinning: basic mechanisms, early history and current status, and an in-depth discussion of various applications (biomedical, environmental, sensors, energy, catalysis, textiles). We aim to provide readers who are currently involved in certain aspects of coaxial electrospinning research an appreciation of other applications and of current results.

Stress Biomarkers in Biological Fluids and Their Point-of-Use Detection.

Hormones produced by glands in the endocrine system and neurotransmitters produced by the nervous system control many bodily functions. The concentrations of these molecules in the body are an indication of its state, hence the use of the term biomarker. Excess concentrations of biomarkers, such as cortisol, serotonin, epinephrine, and dopamine, are released by the body in response to a variety of conditions, for example, emotional state (euphoria, stress) and disease. The development of simple, low-cost modalities for point-of-use (PoU) measurements of biomarkers levels in various bodily fluids (blood, urine, sweat, saliva) as opposed to conventional hospital or lab settings is receiving increasing attention. This paper starts with a review of the basic properties of 12 primary stress-induced biomarkers: origin in the body (i.e., if they are produced as hormones, neurotransmitters, or both), chemical composition, molecular weight (small/medium size molecules and polymers, ranging from ∼100 Da to ∼100 kDa), and hydro- or lipophilic nature. Next is presented a detailed review of the published literature regarding the concentration of these biomarkers found in several bodily fluids that can serve as the medium for determination of the condition of the subject: blood, urine, saliva, sweat, and, to a lesser degree, interstitial tissue fluid. The concentration of various biomarkers in most fluids covers a range of 5-6 orders of magnitude, from hundreds of nanograms per milliliter (∼1 µM) down to a few picograms per milliliter (sub-1 pM). Mechanisms and materials for point-of-use biomarker sensors are summarized, and key properties are reviewed. Next, selected methods for detecting these biomarkers are reviewed, including antibody- and aptamer-based colorimetric assays and electrochemical and optical detection. Illustrative examples from the literature are discussed for each key sensor approach. Finally, the review outlines key challenges of the field and provides a look ahead to future prospects.

Absorption of Ethylene on Membranes Containing Potassium Permanganate Loaded into Alumina-Nanoparticle-Incorporated Alumina/Carbon Nanofibers.

Ethylene is a natural aging hormone in plants, and controlling its concentration has long been a subject of research aimed at reducing wastage during packaging, transport, and storage. We report on packaging membranes, produced by electrospinning, that act as efficient carriers for potassium permanganate (PPM), a widely used ethylene oxidant. PPM salt loaded on membranes composed of alumina nanofibers incorporating alumina nanoparticles outperform other absorber systems and oxidize up to 73% of ethylene within 25 min. Membrane absorption of ethylene generated by avocados was totally quenched in 21 h, and a nearly zero ethylene concentration was observed for more than 5 days. By comparison, the control experiments exhibited a concentration of 53% of the initial value after 21 h and 31% on day 5. A high surface area of the alumina nanofiber membranes provides high capacity for ethylene absorption over a long period of time. In combination with other properties, such as planar form, flexibility, ease of handling, and lightweight, these membranes are a highly desirable component of packaging materials engineered to enhance product lifetime.

Engineering a simple lateral flow device for animal blood coagulation monitoring.

Increasing numbers of animals are diagnosed with thromboembolism, requiring anticoagulation treatment to prevent thrombotic events. Frequent and periodic coagulation monitoring is critical to ensure treatment effectiveness and patient safety by limiting blood coagulation ability within the desired therapeutic range. Point-of-care diagnostics is an ideal candidate for frequent coagulation monitoring due to rapid test results and no need for laboratory setting. This article reports the first utilization of no-reaction lateral flow assay (nrLFA) device for simple and low-cost animal blood coagulation monitoring in resource-limited setting. The nrLFA device consists of sample pad, analytical membrane and wicking pad, without conjugate pad, reagent printing or membrane drying. Citrated and heparinized animal blood were utilized to mimic different blood coagulation abilities in vitro by adding reversal agents CaCl2 and protamine sulfate. The travel distance of red blood cells (RBCs) on the nrLFA after a pre-determined test time serves as endpoint marker. Upon adding 500 mM CaCl2 solution to citrated bovine, canine, rabbit and equine blood, the average travel distance decreases from 10.9 to 9.4 mm, 8.8 to 5.7 mm, 12.6 to 9 mm, and 15.3 to 11.3 mm, respectively. For heparinized bovine and rabbit blood, the average distance decreases from 14.5 to 11.4 mm and from 9.8 to 7.2 mm, respectively, when adding 300 mg/l protamine sulfate solution. The effect of hematocrit on RBC travel distance in the nrLFA was also investigated. The nrLFA device will potentially improve treatment efficiency, patient safety, quality of life, and satisfaction for both animal patients and their owners.

Selective pH-Responsive Core-Sheath Nanofiber Membranes for Chem/Bio/Med Applications: Targeted Delivery of Functional Molecules.

Core-sheath fibers using different Eudragit materials were successfully produced, and their controlled multi-pH responses have been demonstrated. Core-sheath fibers made of Eudragit L 100 (EL100) core and Eudragit S 100 (ES100) sheath provide protection and/or controlled release of core material at pH 6 by adjusting the sheath thickness (controlled by the flow rate of source polymer solution). The thickest sheath (∼250 nm) provides the least core release ∼1.25%/h, while the thinnest sheath (∼140 nm) provides much quicker release ∼16.75%/h. Furthermore, switching core and sheath material dramatically altered the pH response. Core-sheath fibers made of ES100 core and EL100 sheath can provide a consistent core release rate, while the sheath release rate becomes higher as the sheath layer becomes thinner. For example, the thinnest sheath (∼120 nm) provides a core and sheath release ratio of 1:2.5, while the thickest sheath (∼200 nm) shows only a ratio of 1:1.7. All core-sheath Eudragit fibers show no noticeable release at pH 5, while they are completely dissolved at pH 7. Extremely high surface area in the porous network of the fiber membranes provides much faster (>30 times) response to external pH changes as compared to that of equivalent cast films.

Point-of-care coagulation monitoring: first clinical experience using a paper-based lateral flow diagnostic device.

Vitamin K antagonists such as warfarin are the most widely used class of oral anticoagulants. Due to a narrow therapeutic window, patients on warfarin require regular monitoring. Self-testing using point-of-care (POC) diagnostic devices is available, but cost makes this monitoring method beyond reach for many. The main objective of this research was to assess the clinical utility of a low-cost, paper-based lateral flow POC diagnostic device developed for anticoagulation monitoring without the need for a separate electronic reader. Custom-fabricated lateral flow assay (LFA) test strips comprised of a glass fiber sample pad, a nitrocellulose analytical membrane, a cellulose wicking pad, and a plastic backing card were assembled in a plastic cassette. Healthy volunteers and patients on warfarin therapy were recruited for this prospective study. For each participant, a whole blood sample was collected via fingerstick to determine: (1) international normalized ratio (INR) using the CoaguChek® XS coagulometer, (2) hematocrit by centrifugation, and (3) red blood cell (RBC) travel distance on the experimental LFA device after 240 s using digital image analysis. RBC travel distance measured on the LFA device using blood samples obtained from warfarin patients positively correlated with increasing INR value and the LFA device had the capability to statistically distinguish between healthy volunteer INR values and those for patients groups with INR ≥ 2.6. From these data, it is predicted that this low-cost, paper-based LFA device can have clinical utility for identifying anticoagulated patients taking vitamin K antagonists who are outside of the desired therapeutic efficacy window.

Stimuli-Responsive Self-Immolative Polymer Nanofiber Membranes Formed by Coaxial Electrospinning.

The first self-immolative polymer (SIP) nanofiber membrane is demonstrated in this report, in which the immolation can be triggered by external stimulus. Electrospun SIP/polyacrylonitrile (PAN) fibers provide depolymerization that is ∼25 times quicker and more responsive (i.e., immolation) than that of a cast film in the triggering condition. Depolymerization of SIP in the SIP/PAN blended fiber membrane results in the transition of the surface properties from hydrophobic (∼110°) to hygroscopic (∼0°). Triggered release of encapsulated functional molecules was demonstrated using coaxially electrospun fiber membrane made of a SIP/PAN blend sheath and polyvinylpyrrolidone/dye core. Coaxial fibers with the SIP/PAN sheath provide minimal release of the encapsulated material in nontriggering solution, while it releases the encapsulated material instantly when the triggering condition is met. Its versatility has been strengthened compared to that of non-SIP coaxial fibers that provide no triggering reaction by external stimulus.

Long-term antimicrobial effect of nisin released from electrospun triaxial fiber membranes.

Electrospun membranes encapsulating nisin in the core of multi-layer coaxial fibers, with a hydrophobic PCL intermediate layer and a hygroscopic cellulose acetate sheath, have been demonstrated to provide long-term antimicrobial activity combined with a hygroscopic outer layer. Antimicrobial performance has been evaluated using modified versions of the antimicrobial textile test AATCC 100 and AATCC 147 against Staphylococcus aureus. The AATCC 147 tests indicate that antimicrobial activity persists up to 7days. The quantitative analysis from the AATCC 100 test indicates that tri-layer coaxial ("triaxial") electrospun fiber membranes provide >99.99% bacteria kill (4logkill) for up to five days. This indicates that the nisin-incorporated triaxial fibers have excellent biocidal activities for up to 5days and then provide biostatic activity for 2 or more days. Compared with other types of electrospun membranes, such as core-sheath coaxial ("coaxial") and single homogenous fibers, triaxial fiber membranes provided more robust and more sustained antimicrobial activity. Single fibers with nisin showed relatively weak activity and only for one day. Coaxial fiber membranes exhibited antimicrobial activity for a long period, but their biocidal activity was much weaker than that of triaxial fiber membranes, and only exhibited >99% bacteria kill (2logkill) after 1day of exposure. STATEMENT OF SIGNIFICANCE: The increase in drug resistant pathogens has driven the need for alternative treatments that are effective against resistant bacteria and do not contribute to drug resistance. Nisin is an excellent model bacteriocin for antimicrobials because of its size and mode of action, and has been extensively used as FDA-approved food preservatives without any problematic resistance growth in bacteria during past decades. Nisin-containing fibers have been previously reported using conventional electrospinning but sustained antimicrobial effect has not been obtained. Here, we report the encapsulation of nisin into a multi-layered nanofiber construct using triaxial electrospinning in order to obtain a long-term antimicrobial activity. This will be highly beneficial in many applications, such as protective textiles, food packaging and cancer therapy.

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers.

Magnetic beads with ~1.9 µm average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 µl/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

Electrospun carbon nanofiber modified electrodes for stripping voltammetry.

Electrospun polyacrylonitrile (PAN) based carbon nanofibers (CNFs) have attracted intense attention due to their easy processing, high carbon yield, and robust mechanical properties. In this work, a CNF modified glassy carbon (GC) electrode that was coated with Nafion polymer was evaluated as a new electrode material for the simultaneous determination of trace levels of heavy metal ions by anodic stripping voltammetry (ASV). Pb(2+) and Cd(2+) were used as a representative system for this initial study. Well-defined stripping voltammograms were obtained when Pb(2+) and Cd(2+) were determined individually and then simultaneously in a mixture. Compared to a bare GC electrode, the CNF/Nafion modified GC (CNF/Nafion/GC) electrode improved the sensitivity for lead detection by 8-fold. The interface properties of the CNF/Nafion/GC were characterized by electrochemical impedance spectroscopy (EIS), which showed the importance of the ratio of CNF/Nafion on electrode performance. Under optimized conditions, the detection limits are 0.9 and 1.5 nM for Pb(2+) and Cd(2+), respectively.