On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10³ and 1.1 × 10³ CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.     

Separation and concentration of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> sporangia by inertial focusing in curving microfluidic flows

We present an integrated microfluidic system for performing isolation and concentration of Phytophthora ramorum pathogens using a chip whose working principle is based on inertial lateral migration in curving flows. The chip was fabricated from multiple layers of thermoplastic polymers and features an embedded spiral separation channel along with peristaltic microvalves for fluidic operation and process control. A pumping system paired with a fully programmable pressure manifold is used to boost concentration levels by recirculating the sample liquid multiple times through the separation chip, making it possible to reduce sample volumes from 10 to 1 mL or less. The system was calibrated using fluorescent polymer particles with a nominal diameter of 30 µm which is comparable to that of P. ramorum sporangia. The separation process has been shown to be highly effective and more than 99% of the beads can be recovered in the concentrated batch. Experiments conducted with P. ramorum sporangia have shown that a 5.3-fold increase in pathogen content with 95% recovery can be achieved using three subsequent concentration cycles. The utility of the method has been validated by processing a sample derived from infested Rhododendron leaves where a 6.1-fold increase in the concentration of P. ramorum has been obtained after four concentration cycles. Although specifically designed and demonstrated for sporangia of P. ramorum, the method and related design rules can easily be extended to other microbial organisms, effectively supporting bioanalytical applications where efficient, high-throughput separation of target species is of primary concern.     

Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via online pre-concentration of field-amplified sample stacking

In this paper, polydopamine/gold nanoparticles (PDA/Au NPs) were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis (MCE) for the separation of reduced glutathione (GSH) and oxidized glutathione (GSSH). The formation of the PDA/Au NPs was characterized by scanning electron microscopy, transmission electron microscope, UV–Vis spectra and ATR-FTIR. An online pre-concentration strategy involving field-amplified sample stacking was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria (Escherichia coli, Staphylococcus aureus and Salmonella enterica serovar Typhimurium) and HaCaT cells samples by MCE with laser-induced fluorescence detection. The influences of the separation voltage, the concentration of the running buffer and the pH value of running buffer, were carefully investigated. Using this studied method, GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s. The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM, respectively (S/N = 3), which corresponded to approximately 180–301-fold improvements in peak height. Moreover, this method was successfully applied to the analysis of bacteria (E. coli, S. aureus and S. typhimurium) and HaCaT cell samples with a satisfactory recovery rate.     

Anomalous pulse change in gravity-driven microfluidic oscillator and its application to photodiode switching

Electrofluidic analogy is useful because it provides a method to significantly reduce the reliance of microfluidic chips on dynamic off-chip controllers. Among the functions developed by the analogy, conversion from constant to pulsatile pressure is critical and is yet to be studied. Here, unlike its counterpart electrical oscillator generating square pulses more slowly with decreasing the input voltage, we report that a microfluidic oscillator generates sawtooth pressure pulses more rapidly with decreasing the input pressure (P_I) at 1–2 kPa. Further, with decreasing P_I, the oscillator generates square pulses at P_I > 3.4 kPa, but its operation unexpectedly stops at 2.1 < P_I < 3.4 kPa. We analyze its underlying mechanism with a sophisticated model including a dynamic interaction of the oscillator components and reveal the critical role of the dynamic property of oscillator valves. Additionally, we show electrofluidic switching of a photodiode with the oscillator. The understanding obtained in this study would be essential for developing microfluidic circuits using electrofluidic analogy.     

Star reduction among minimal length addition chains

An addition chain for a natural number n is a sequence \({1=a_0 < a_1 < \cdots < a_r=n}\) of numbers such that for each 0 < i ≤ r, a _i  = a _j  + a _k for some 0 ≤ k ≤ j < i. The minimal length of an addition chain for n is denoted by ?(n). If j = i ? 1, then step i is called a star step. We show that there is a minimal length addition chain for n such that the last four steps are stars. Then we conjecture that there is a minimal length addition chain for n such that the last \({\lfloor\frac{\ell(n)}{2}\rfloor}\)-steps are stars. We verify that the conjecture is true for all numbers up to 2¹⁸. An application of the result and the conjecture to generate a minimal length addition chain reduce the average CPU time by 23–29% and 38–58% respectively, and memory storage by 16–18% and 26–45% respectively for m-bit numbers with 14 ≤ m ≤ 22.     

Threshold quantum secret sharing based on single qubit

Based on unitary phase shift operation on single qubit in association with Shamir’s (t, n) secret sharing, a (t, n) threshold quantum secret sharing scheme (or (t, n)-QSS) is proposed to share both classical information and quantum states. The scheme uses decoy photons to prevent eavesdropping and employs the secret in Shamir’s scheme as the private value to guarantee the correctness of secret reconstruction. Analyses show it is resistant to typical intercept-and-resend attack, entangle-and-measure attack and participant attacks such as entanglement swapping attack. Moreover, it is easier to realize in physic and more practical in applications when compared with related ones. By the method in our scheme, new (t, n)-QSS schemes can be easily constructed using other classical (t, n) secret sharing.     

Isolation of magnetically tagged cancer cells through an integrated magnetofluidic device

Circulating tumour cells (CTC) in the bloodstream has been implicated in cancer metastasis. Efficient removal of CTC could potentially be an effective therapeutic measure against cancer metastasis. In this study, the hydrodynamic focusing flow in microfluidic channels (R _e  ? 1) was considered together with the magnetophoretic force. The localised magnetic field was achieved through a passivated current-carrying multilayered microstripline, where the generated field gradient was used to attract the magnetic beads to the desired outlet. The experimental results show that the device is capable of isolating purely magnetic beads with an efficiency of 91 % while isolation efficiency of the magnetically tagged HeLa cervical cancer cells from cell suspension yielded an isolation efficiency of 79 %.     

A linear-time algorithm for finding Hamiltonian (<Emphasis Type="Italic">s</Emphasis>, <Emphasis Type="Italic">t</Emphasis>)-paths in odd-sized rectangular grid graphs with a rectangular hole

A grid graph \(G_{\mathrm{g}}\) is a finite vertex-induced subgraph of the two-dimensional integer grid \(G^\infty \). A rectangular grid graph R(m, n) is a grid graph with horizontal size m and vertical size n. A rectangular grid graph with a rectangular hole is a rectangular grid graph R(m, n) such that a rectangular grid subgraph R(k, l) is removed from it. The Hamiltonian path problem for general grid graphs is NP-complete. In this paper, we give necessary conditions for the existence of a Hamiltonian path between two given vertices in an odd-sized rectangular grid graph with a rectangular hole. In addition, we show that how such paths can be computed in linear time.     

Operating regimes of a magnetic split-flow thin (SPLITT) fractionation microfluidic device for immunomagnetic separation

Magnetic bead-based immunoassays in the microfluidic format have attracted particular interest as it has several advantages over other microfluidic separation techniques. Magnetic split-flow thin fractionation (SPLITT) is a compact version of microfluidic sorting where a bidispersed or polydispersed suspension of magnetic particle–analyte conjugates can be selectively isolated into co-flowing streams of nearly monodispersed particles. Although the device offers capability of identifying and separating more than one target analytes simultaneously, its performance is sensitive to the slightest variation of the operating condition. Herein, we have numerically investigated the performance of a microscale magnetic SPLITT device. Using a coupled Eulerian–Lagrangian approach, we have evaluated the capture efficiency (CE) and separation index (SI) for each particle type collected at their designated outlet of the SPLITT device and identified the best regimes of operating parameters. While the CE figures are found to be best represented by a group variable Π, the SI values are better represented as function of the product of the group variables γ and β; the SI versus Π plots clearly separate into two basic trends: one for constant β (i.e., varying γ) and the other for constant γ (i.e., varying β). Our study prescribes the desired operating regimes of a microfluidic magnetophoretic SPLITT device in a practical immunomagnetic separation application.     

An orthogonal analysis method for decoupling the nozzle geometrical parameters of microthrusters

This paper proposes an orthogonal analysis method for decoupling the multiple nozzle geometrical parameters of microthrusters, thus an reconfigured design can be implemented to generate a proper thrust. In this method, the effects of various nozzle geometrical parameters, including throat width W _t , half convergence angle θ _in , half divergence angle θ _out , exit-to-throat section ratio W _e /W _t and throat radius of the curvature R _t /W _t , on the performance of microthrusters are sorted by range analysis. Analysis results show that throat width seriously affects thrust because range value of 67.53 mN is extremely larger than the range value of other geometry parameters. For average specific impulse (ASI), the range value of exit-to-throat section ratio W _e /W _t and half divergence angle θ _out are 4.82 s and 3.72 s, respectively. Half convergence angle with the range value of 0.39 s and throat radius with 0.32 s have less influence on ASI compared with exit-to-throat section ratio and half divergence angle. When increasing the half convergence angle from 10° to 40° and throat radius of the curvature from 3 to 9, average specific impulse initially decreases and then increases. A MEMS solid propellant thruster (MSPT) with the reconfigured geometrical parameters of nozzle is fabricated to verify the feasibility of the proposed method. The thrust of the microthruster can reach 25 mN. Power is estimated to be 0.84 W. This work provides design guideline to reasonably configure geometry parameters of microthruster.     

On the Price of Heterogeneity in Parallel Systems

Suppose we have a parallel or distributed system whose nodes have limited capacities, such as processing speed, bandwidth, memory, or disk space. How does the performance of the system depend on the amount of heterogeneity of its capacity distribution? We propose a general framework to quantify the worst-case effect of increasing heterogeneity in models of parallel systems. Given a cost function g(C,W) representing the system’s performance as a function of its nodes’ capacities C and workload W (such as the makespan of an optimum schedule of jobs W on machines C), we say that g has price of heterogeneity α when for any workload, cost cannot increase by more than a factor α if node capacities become arbitrarily more heterogeneous. The price of heterogeneity also upper bounds the “value of parallelism”: the maximum benefit obtained by increasing parallelism at the expense of decreasing processor speed. We give constant or logarithmic bounds on the price of heterogeneity of several well-known job scheduling and graph degree/diameter problems, indicating that in many cases, increasing heterogeneity can never be much of a disadvantage.     

Negation-Limited Complexity of Parity and Inverters

In negation-limited complexity, one considers circuits with a limited number of NOT gates, being motivated by the gap in our understanding of monotone versus general circuit complexity, and hoping to better understand the power of NOT gates. We give improved lower bounds for the size (the number of AND/OR/NOT) of negation-limited circuits computing Parity and for the size of negation-limited inverters. An inverter is a circuit with inputs x ₁,…,x _n and outputs ¬ x ₁,…,¬ x _n . We show that: (a) for n=2 ^r ?1, circuits computing Parity with r?1 NOT gates have size at least 6n?log?₂(n+1)?O(1), and (b) for n=2 ^r ?1, inverters with r NOT gates have size at least 8n?log?₂(n+1)?O(1). We derive our bounds above by considering the minimum size of a circuit with at most r NOT gates that computes Parity for sorted inputs x ₁≥???≥x _n . For an arbitrary r, we completely determine the minimum size. It is 2n?r?2 for odd n and 2n?r?1 for even n for ?log?₂(n+1)??1≤r≤n/2, and it is ?3n/2??1 for r≥n/2. We also determine the minimum size of an inverter for sorted inputs with at most r NOT gates. It is 4n?3r for ?log?₂(n+1)?≤r≤n. In particular, the negation-limited inverter for sorted inputs due to Fischer, which is a core component in all the known constructions of negation-limited inverters, is shown to have the minimum possible size. Our fairly simple lower bound proofs use gate elimination arguments in a somewhat novel way.     

A new strategy for generating shortest addition sequences

An addition sequence problem is given a set of numbers X = {n ₁, n ₂, . . . , n _m }, what is the minimal number of additions needed to compute all m numbers starting from 1? This problem is NP-complete. In this paper, we present a branch and bound algorithm to generate an addition sequence with a minimal number of elements for a set X by using a new strategy. Then we improve the generation by generalizing some results on addition chains (m = 1) to addition sequences and finding what we will call a presumed upper bound for each n _j , 1 ≤ j ≤ m, in the search tree.     

Deterministic lateral displacement (DLD) in the high Reynolds number regime: high-throughput and dynamic separation characteristics

Recent progress in the development of biosensors has created a demand for high-throughput sample preparation techniques that can be easily integrated into microfluidic or lab-on-a-chip platforms. One mechanism that may satisfy this demand is deterministic lateral displacement (DLD), which uses hydrodynamic forces to separate particles based on size. Numerous medically relevant cellular organisms, such as circulating tumor cells (10–15 µm) and red blood cells (6–8 µm), can be manipulated using microscale DLD devices. In general, these often-viscous samples require some form of dilution or other treatment prior to microfluidic transport, further increasing the need for high-throughput operation to compensate for the increased sample volume. However, high-throughput DLD devices will require a high flow rate, leading to an increase in Reynolds numbers (Re) much higher than those covered by existing studies for microscale (≤?100 µm) DLD devices. This study characterizes the separation performance for microscale DLD devices in the high-Re regime (10?<?Re?<?60) through numerical simulation and experimental validation. As Re increases, streamlines evolve and microvortices emerge in the wake of the pillars, resulting in a particle trajectory shift within the DLD array. This differs from previous DLD works, in that traditional models only account for streamlines that are characteristic of low-Re flow, with no consideration for the transformation of these streamlines with increasing Re. We have established a trend through numerical modeling, which agrees with our experimental findings, to serve as a guideline for microscale DLD performance in the high-Re regime. Finally, this new phenomenon could be exploited to design passive DLD devices with a dynamic separation range, controlled simply by adjusting the device flow rate.     

Hydrodynamic focusing and interdistance control of particle-laden flow for microflow cytometry

Single-file focusing and minimum interdistance of micron-size objects in a sample is a prerequisite for accurate flow cytometry measurements. Here, we report analytical models for predicting the focused width of a sample stream b as a function of channel aspect ratio α, sheath-to-sample flow rate ratio f and viscosity ratio λ in both 2D and 3D focusing. We present another analytical model to predict spacing between an adjacent pair of objects in a focused sample stream as a function of sample concentration C, mobility ? of the objects in the prefocused and postfocused regions and flow rate ratio f in both 2D and 3D flow focusing. Numerical simulations are performed using Ansys Fluent VOF model to predict the width of sample stream in 2D and 3D hydrodynamic focusing for different sample-to-sheath viscosity ratios, aspect ratios and flow rate ratios. Experiments are performed on both planar and three-dimensional devices fabricated in PDMS to demonstrate focusing of sample stream and spacing of polystyrene beads in the unfocused and focused stream at different sample concentrations C. The predictions of the analytical model and simulations are compared with experimental data, and a good match is found (within 12 %). Further, mobility of objects is experimentally studied in 2D and 3D focusing, and the spread of the mobility data is used as tool for the demonstration of particle focusing in flow cytometer applications.     

Tilings of nonorientable surfaces by Steiner triple systems

A Steiner triple system of order n (for short, STS(n)) is a system of three-element blocks (triples) of elements of an n-set such that each unordered pair of elements occurs in precisely one triple. Assign to each triple (i,j,k) ? STS(n) a topological triangle with vertices i, j, and k. Gluing together like sides of the triangles that correspond to a pair of disjoint STS(n) of a special form yields a black-and-white tiling of some closed surface. For each n ≡ 3 (mod 6) we prove that there exist nonisomorphic tilings of nonorientable surfaces by pairs of Steiner triple systems of order n. We also show that for half of the values n ≡ 1 (mod 6) there are nonisomorphic tilings of nonorientable closed surfaces.     

Restricted (<Emphasis Type="Italic">k</Emphasis>, <Emphasis Type="Italic">n</Emphasis>)-threshold quantum secret sharing scheme based on local distinguishability of orthogonal multiqudit entangled states

In this work, we study a restricted (k, n)-threshold access structure. According to this structure, we construct a group of orthogonal multipartite entangled states in d-dimensional system and investigate the distinguishability of these entangled states under restricted local operations and classical communication. Based on these properties, we propose a restricted (k, n)-threshold quantum secret sharing scheme (called LOCC-QSS scheme). The k cooperating players in the restricted threshold scheme come from all disjoint groups. In the proposed protocol, the participants distinguish these orthogonal states by the computational basis measurement and classical communication to reconstruct the original secret. Furthermore, we also analyze the security of our scheme in three primary quantum attacks and give a simple encoding method in order to better prevent the participant conspiracy attack.     

Developing heatable microfluidic chip to generate gelatin emulsions and microcapsules

The heatable microfluidic chip developed herein successfully integrates a microheater and flow-focusing device to generate uniform-sized gelatin emulsions under various flow rate ratios (sample phase/oil phase, Q _s/Q _o) and driven voltages. The gelatin emulsions can be applied to encapsulate vitamin C for drug release. Our goal is to create the thermal conditions for thermo-sensitive hydrogel materials in the microfluidic chip and generate continuous and uniform emulsions under any external environment. The gelatin emulsion sizes have a coefficient of variation of <5 % and can be precisely controlled by altering the flow rate ratio (Q _s/Q _o) and driven voltage. The gelatin emulsion diameters range from 45 to 120 μm. Moreover, various sizes of these gelatin microcapsules containing vitamin C were used for drug release. The developed microfluidic chip has the advantages of a heatable platform in the fluid device, active control over the emulsion diameter, the generation of uniform-sized emulsions, and simplicity. This new approach for gelatin microcapsules will provide many potential applications in drug delivery and pharmaceuticals.     

The weight-constrained maximum-density subtree problem and related problems in trees

Given a tree T=(V,E) of n nodes such that each node v is associated with a value-weight pair (val _v ,w _v ), where value val _v is a real number and weight w _v is a non-negative integer, the density of T is defined as \(\frac{\sum_{v\in V}{\mathit{val}}_{v}}{\sum_{v\in V}w_{v}}\). A subtree of T is a connected subgraph (V′,E′) of T, where V′?V and E′?E. Given two integers w _min? and w _max?, the weight-constrained maximum-density subtree problem on T is to find a maximum-density subtree T′=(V′,E′) satisfying w _min?≤∑_v∈V′ w _v ≤w _max?. In this paper, we first present an O(w _max? n)-time algorithm to find a weight-constrained maximum-density path in a tree T, and then present an O(w _max? ² n)-time algorithm to find a weight-constrained maximum-density subtree in T. Finally, given a node subset S?V, we also present an O(w _max? ² n)-time algorithm to find a weight-constrained maximum-density subtree in T which covers all the nodes in S.     

Almost cover-free codes

We say that an s-subset of codewords of a code X is (s, l)-bad if X contains l other codewords such that the conjunction of these l words is covered by the disjunction of the words of the s-subset. Otherwise, an s-subset of codewords of X is said to be (s, l)-bad. A binary code X is called a disjunctive (s, l) cover-free (CF) code if X does not contain (s, l)-bad subsets. We consider a probabilistic generalization of (s, l) CF codes: we say that a binary code is an (s, l) almost cover-free (ACF) code if almost all s-subsets of its codewords are (s, l)-good. The most interesting result is the proof of a lower and an upper bound for the capacity of (s, l) ACF codes; the ratio of these bounds tends as s→∞ to the limit value log₂ e/(le).