Sheath-enhanced concentration and on-chip detection of bacteria from an extremely low-concentration level

Abstract

Microfluidic-based sheath flow focusing methods have been widely used for efficiently isolating, concentrating, and detecting pathogenic bacteria for various biomedical applications due to their enhanced sensitivity and exceptional integration. However, such a microfluidic device usually needs complicated device fabrication and sample dilution, hampering the efficient and sensitive identification of target bacteria. In this study, we develop and fabricate a sheath-assisted and pneumatic-induced nano-sieve device for achieving the improved on-chip concentration and sensitive detection of Staphylococcus aureus (MRSA). The optimized nanochannel design with diverging configuration is beneficial to the regulation of the hydrodynamic flow while the sheath flow is focusing the sample to the confined region as expected. Per the experimental finding, a high flow ratio (sheath flow/sample flow) presents enhanced target concentration by comparing with a low flow ratio. With this setup, MRSA bacteria with an extremely low concentration of ∼100 CFU mL⁻¹ are successfully and sensitively detected under a fluorescence microscope, less than 30 min, demonstrating a reliable sheath-enhanced concentration and on-chip detection for target bacteria. Additionally, the theoretical model introduced here further rationalizes the working principle of our nano-sieve device, potentially guiding the optimization of next generation devices for highly sensitive and accurate on-chip bacteria detection at a much lower concentration level below 100 CFU mL⁻¹.

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Introduction

The efficient detection and identification of pathogenic bacteria have become a critical concern in various fields including clinical diagnostics,^1,2 food safety,^3,4 and environmental monitoring.^4,5 For example, methicillin-resistant Staphylococcus aureus (MRSA) can cause a range of infections from mild skin infections to some severe diseases, including pneumonia^6,7 and bloodstream infections.^8,9 Conventional detection methods require tedious steps including pre-enrichment of target bacteria from low population to high population, selective enrichment of media or conditions, and biochemical screening.¹⁰ The whole process presents a complicated and lengthy series of tests for the results, which usually need 2 or 3 days or even longer.^11,12 Recently, innovative technologies for detecting pathogenic bacteria, such as flow cytometry,^13,14 enzyme-linked immunosorbent assay (ELISA),^15,16 and polymerase chain reaction (PCR),^17,18 have been explored due to their shorter processing times, higher sensitivity, and specificity. However, these technologies often rely on specialized training for complicated and delicate operations.¹⁹ Hence, establishing a reliable and cost-effective platform could be essential for rapidly and sensitively detecting pathogenic bacteria with a simple on-chip operation.

Over the past few years, microfluidic-based platforms have been powerful analytical and diagnostic tools for various biomedical applications, including sample enrichment, separation, and detection of target bacteria.¹⁹ Among these applications, microfluidic-based sheath-assisted flow focusing could regulate the micro/nanoparticle suspension, hence presenting the enhancement of significantly concentrated sample solutions^20,21 and sensitively detecting target bacteria.^22,23 As an example, Rodríguez-Lorenzo et al. reported that a flow-focusing microfluidic device could couple with surface-enhanced Raman scattering (SERS) for on-chip bacteria concentration and fast detection by using gold nano-stars as SERS tags.²⁴ This system enables distinguishing two different types of bacteria under in-flow detection. Yamaguchi et al. employed a sheath-assisted microfluidic device to achieve on-chip detection of bacterial cells from sample solution. This method offers a fast (∼1.5 h) in situ detection and on-site monitoring of fluorescence-stained bacteria in the desired region through sheath flow alignment.²⁵ However, such a sheath flow-based microfluidic platform usually requires extra fluids for sheath flow conditions, therefore diluting the initial samples,²⁶ which is not suitable for samples with low-concentration targets.

Our team presented a pneumatic-induced and bead-stacked nano-sieve device, enabling efficient purification of drug-resistant bacteria (e.g., MRSA) from human plasma samples and achieving a fast and sensitive detection by coupling with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)–Cas assay.²⁷ Using this approach, however, the target bacteria need to be retrieved from a wide nano-sieve channel, which always results in unwanted loss of target bacteria, especially when dealing with bacteria samples at low-concentration levels.

To address these limitations, in this study, we aim to develop a reliable microfluidic device enhanced by sheath flow focusing for rapid and sensitive detection of MRSA bacteria with extremely low-concentration levels (Fig. 1a). We, for the first time, introduced a straightforward and cost-effective fabrication method to create a sheath-assisted and pneumatic-induced nano-sieve device, avoiding the complicated and costly micro and nanofabrication techniques that typically involve labor-intensive nanolithography. Secondly, the specially designed diverging channel allows for a linearly decreasing flow velocity along the central flow axis.²⁸ Plus, two sheath flow channels were incorporated to focus and narrow the mainstream fluid under adjustable and stable flow conditions. Additionally, optimized stacked beads within the nano-sieve channel further enhance the system's functionality by physically concentrating the target bacteria from the applied sample and isolating waste fluids. Consequently, this established system enables precise flow control, effectively capturing the target bacteria in a confined region. Thirdly, the pneumatic chamber positioned above the nano-sieve channel stabilizes the stacked components by counterbalancing the hydrodynamic pressure of the flowing stream.²⁹ In addition, our physical models validate and rationalize the working principles of this nano-sieve system. Under a fluorescence microscope, those MRSA bacteria can be eventually enriched in a locally confined region and easily distinguished from sample solutions with various concentrations, down to a low level of ∼100 CFU mL⁻¹. To evaluate the capacity of the nano-sieve system, fluorescent nanoparticles with a diameter of 0.5 μm were selected in this study, due to the strong fluorescence intensity for distinguishable results and highly similar morphology to MRSA bacteria with a diameter of 0.5–0.7 μm.³⁰ The experimental results indicate the stable accumulation of small-sized nanoparticle targets and the promising concentration of MRSA bacteria in a predefined and desired region, demonstrating a robust and reliable nano-sieve system potentially for biomedical applications regarding on-chip detection of infectious pathogens, such as sepsis diagnostics.^9,17

Fig. 1 (a-i) The schematic of the sheath-assisted and pneumatic-induced nano-sieve device presenting an overview of channel configuration. (a-ii) The local region of MRSA–bead interface. (a-iii) MRSA physically concentrated by stacked beads with diameters of 10 μm and 1 μm. (b) Fabrication procedure of the device by plasma bonding the PDMS pneumatic chamber, PDMS thin film, and channel-patterned glass substrate. (c) The optical picture showing the experimental device with low and high magnifications.

Experimental methods

Nano-sieve channel design

The main channel features tapered geometry with a diverging configuration, hence regulating the flow behaviour with a linearly decreased flow velocity along the flow centreline.²⁸ Moreover, two sheath flow channels are designed to focus and narrow the mainstream of fluids from the central inlet, aiming to create a specific range of areas for concentrating the applied target particles, while isolating the waste fluids through the predefined stacked beads.^31,32 This specifically designed diverging nano-sieve channel could better enable the flow behaviour at the interface of stacked beads when the sheath flow focusing method is applied. The nano-sieve channel has a constant height of 200 nm, a total length of 14 mm, and a width of tapered channels from 1.5 mm at the central inlet to 6.5 mm at the outlet. The side channel for the sheath flow has a width of 2 mm. Above the nano-sieve channel, a pneumatic chamber (2 mm in height and 2 mm in width) is also equipped to counterbalance the hydrodynamic pressure from the continuous flowing fluids within the nano-sieve channel,²⁹ and to secure the configuration of stacked beads for efficiently capturing and concentrating the applied particles.^31,32

Nano-sieve fabrication

A thin layer of tetraethyl orthosilicate (TEOS) was deposited on a cleaned 4-inch glass wafer (D263 glass, UniversityWafer, Inc.) with plasma-enhanced chemical vapor deposition (PECVD, Unaxis 790). Then, a layer of positive photoresist (S1813) with a thickness of ∼1.5 μm was coated on the TEOS layer by using a spin-coater. A mask aligner was applied to transfer the specific patterns from a plastic photomask (Fineline Imaging) onto the photoresist layer. After UV exposure for ∼9 s, an AZ 300 MIF developer was used to remove the coated photoresist exposed to UV light. Next, a buffered oxide etchant (BOE) (6 : 1) with the surfactant was applied to define the nano-sieve channel with TEOS. The BOE etching rate for TEOS is about 86–93 nm min⁻¹ at room temperature (∼21 °C), which is almost 4 times faster than the BOE etching rate for a glass substrate.³³ After cleaning the whole glass wafer with acetone, followed by IPA and DI water rinsing and air drying, the glass wafer was placed on a hotplate (110 °C, ∼3 min) for the dehydration before spin-coating a sacrificial positive photoresist layer. Finally, each nano-sieve channel was filled with the photoresist to avoid the polydimethylsiloxane (PDMS) collapse during the PDMS–glass bonding process.³⁴

Pneumatic chamber fabrication

A three-dimensional (3D) printed mold (Fictiv) was employed for producing the PDMS replica with the pneumatic chamber (Fig. S1 in the ESI†). The PDMS base and the curing agent (SYLGARD 184 silicone elastomer) were mixed with a weight ratio of 10 : 1, followed by the curing process in an oven at 65 °C overnight. The PDMS thin film of ∼200 μm in thickness was created by using a glass-sandwich approach.²⁷ Then, the cured thin film and pneumatic chamber were bonded together by applying the surface plasma treatment (Electro-Technic Products). The whole piece was baked on a hot plate at 100 °C for 2 h to obtain strong bonding strength. Finally, the nano-sieve channel substrate was bonded with the pneumatic layer through the surface plasma treatment baked at 100 °C for 2 h and stored for further experiments.

Bacterial culture

The methicillin-resistant Staphylococcus aureus strain (ATCC 43300) was purchased from Fisher Scientific. The bacteria were cultured in a tryptic soy broth medium (MilliporeSigma) and maintained on tryptic soy agar plates. The bacterial culture was incubated overnight at 37 °C with shaking at 200 rpm. Following incubation, 1 mL of the culture was centrifuged at 8000g for 5 min to collect the bacterial pellet. This pellet was then resuspended in 1 mL of phosphate-buffered saline (PBS) and washed twice to remove any residual culture medium. The concentration of the bacterial cells was quantified by counting colony-forming units (CFUs) on standard TSA plates.

Bacterial staining

DAPI stock solution (D1306, Thermo Fisher Scientific) with a volume of 2.1 μL was added to 100 μL DI water as the working solution. Then, this working solution was mixed with 900 μL of PBS-based MRSA solution and vortexed for several seconds to obtain the well-mixed sample solution. Subsequently, MRSA bacteria were incubated at room temperature for ∼20 min by following the manufacturer's instruction. The incubated solution was centrifuged at 8000g for 5 min to remove the extra dye. The stained bacteria were resuspended with 1 mL fresh PBS for further use.

Experimental setup

Three inlet holes and one outlet hole of each nano-sieve channel were punched by using a 1 mm biopsy puncher (INTEGRATM MiltexTM) on the designed pattern. Acetone was applied to completely remove the photoresist coated within the channel, followed by the IPA and DI washing process, to prepare a cleaned and clear channel for experiments. The microfluidic tubing (Scientific Commodities Inc., BB31695- PE/3) was applied to connect the needle of a syringe (BD, 1 mL) to the inlet holes and to connect the outlet hole to a centrifuge tube to collect the waste solution from the nano-sieve device. Furthermore, microfluidic tubing was used to connect the pneumatic chamber to a mini air pump (Precigenome LLC) for air regulation during the experiment. One syringe was placed on a single-syringe pump (NE-300, “Just Infusion”) for loading the sample solution. Another two syringes with DI water were placed on a multi-channel syringe pump (SP220I syringe pump) to offer a continuous sheath flow during the experiment. The whole experiment was performed under a fluorescence microscope (Leica DMi8), and monitored by using a digital camera (Leica, K3) and the Leica LAS X software.

Bead stacking configuration

3D microbead stacking was carried out to physically capture and concentrate the target particles from the initial sample solution.³¹ The air pump was first activated at a constant pressure of ∼8 psi. Then, 45 μL of 10 μm magnetic beads with a concentration of 7.64 × 10⁶ beads per mL was injected into the nano-sieve channel by the single-syringe pump at a flow rate of 10 μL min⁻¹, to form a “coarse filter”. Next, 30 μL of 1 μm magnetic beads with a concentration of 7.64 × 10⁹ were introduced to form a “fine filter” at a flow rate of 10 μL min⁻¹. The sheath flow produced at a flow rate of 4 μL min⁻¹ per channel was also applied throughout the whole process of stacked bead configuration.

Focusing stream recognition

The DI water solution mixed with a fluorescence component (10 μM, 5-carboxyfluorescein) was pumped into the nano-sieve with the stacked beads from the central inlet. The flow rate was constantly set at 8 μL min⁻¹. The sheath flow of each channel was gradually tuned from 5 to 15 μL min⁻¹ to observe the profile change of the focusing stream. The width from the minimum neck of the focusing stream was defined as “working width” and measured accordingly by using the “measurement tool” of LAS X software, then recorded for further analysis.

Target particle capture and concentration

After the preparation of stacked beads, 150 μL or 250 μL of sample solutions with fluorescence nanoparticles (Degradex, 2103A, 0.5 μm) with a concentration of 1.71 × 10⁵ beads per mL were injected into the nano-sieve channel at a constant flow rate of 8 μL min⁻¹ and tested by applying a variable flow rate from sheath flow channels. With the same stacked bead setup, 200 μL of stained MRSA bacteria with various concentrations from 100 to 10⁴ CFU mL⁻¹ were tested at flow rates of 8 μL min⁻¹ and 15 μL min⁻¹ for sample solution and sheath flow, respectively. DI water (1 mL) was applied in the sheath flow channels for each test.

Microscope imaging

A digital camera (Leica, K3) mounted on the fluorescence microscope (Leica, DMi8) was used to capture the images for on-chip and off-chip recognition of target particles, including fluorescent beads and stained MRSA bacteria. The GFP filter cube designed for green fluorescence signals and the DAPI filter cube designed for blue fluorescence were selected, depending on the type of applied samples. The sample solution was pipetted onto the glass slide and covered with a cover glass for imaging. The exposure time was set at 500, and the gain value was set at 5, to distinguish the fluorescence signal from applied samples. All images were exported with the same parameter setting for further analysis.

Fluorescence intensity measurement

ImageJ software was used for processing the collected images exported from the LAS X software. The integrated density was measured with the function of “Analyse/Measurement”. Each image was set at the same parameters, such as bright/contrast and selected central area across the channel for measurement, for the quantitative analysis. Each image was at least measured three times.

Theoretical calculation

a. Physical model of determining the width of sample flow. Following the mass conservation, the width of the sample flow, w_m, can be determined by:where Q_m is the flow rate of the sample, u is the average velocity, and H is the height of the channel. Here, we assume the channel has negligible deformation, i.e. H is constant in the experiment. The total flow rate, Q, can then be represented as:

Q = Q_m + 2Q_h = uHw, (2)

where Q_h is one of the flow rates of the two sheath flows and w is the total width of the channel. Substituting eqn (2) into eqn (1) yields:

b. Characterization of the concentration effect. To better understand the mechanism of bead stacking in the nano-sieve, we developed a first-order theoretical model. The concentration of the beads, C, changes over time in the stacking area (∼0.647 mm²) that follows:where k is the accumulating rate of the beads, which can be modeled as k = k₁u^k₂ and k₁ and k₂ are constants. Solving this equation, we have:

C = C₀e^−k₁uk2t (5)

where C₀ is the concentration of the beads pumped into the channel. The deposit of the beads, S, can be modeled as:The solution to this equation is:

S = S₀(1 − e^{−k₁uk₂t}) (7)

Assuming the deposit of the beads is proportional to the observed integrated density fluorescence (IDF), we obtain:

F = αAS₀(1 − e^{−k₁uk₂t}) = F₀(1 − e^{−k₁uk₂t}) (8)

where A is the deposit area and α is the ratio of IDF and the total bead deposit.

Results and discussion

As shown in Fig. 1a, the sample solution with low-concentration of MRSA bacteria is loaded from the central inlet to the bead stacked nano-sieve channel under the pressure-induced pneumatic chamber (Fig. 1a-i). The sheath fluids are introduced from the side channels to pinch the MRSA bacteria suspension flow, therefore focusing the suspended MRSA bacteria physically captured by the predefined stacked magnetic beads with the diameters of 1 μm and 10 μm, at the specific region (Fig. 1a-ii). Meanwhile, the liquid from the sample solution and sheath fluids can be filtered out through the gaps between those stacked beads, and then collected by the waste reservoir. Consequently, the on-chip concentration of MRSA bacteria can be achieved as expected as shown in Fig. 1a-iii. Fig. 1b displays the fabrication process of the proposed nano-sieve device. Briefly, a thin film of TEOS with a thickness of 200 nm was deposited onto a cleaned glass substrate by PECVD. Subsequently, the photoresist layer with designed channel patterns was used as a stencil, while protecting the regions outside the channel patterns. Photolithography is applied for defining the channel pattern from the designed photomask under UV exposure, followed by the development process. Then, BOE wet etching is used for opening the channel area. On the other hand, a layer of PDMS pneumatic chamber is created based on the application of a 3D printed mold, and a PDMS thin film (∼200 μm) was formed via the “sandwiched” approach.²⁷ Finally, the pneumatic chamber, PDMS thin film, and nano-sieve channel substrate are bonded together through the surface plasma treatment, to complete the nano-sieve device. The cross-sectional profile of this nano-sieve device can be found in Fig. S2 (ESI†), which shows that the nano-sieve channel on the glass substrate and the pneumatic chamber as a top layer are separated by the PDMS thin film. The optical pictures in Fig. 1c show the array of practical nano-sieve channels for running experiments. The light-yellow region represents the sacrificial photoresist for assisting the bonding process.

The stacked bead configuration can be realized as shown in Fig. 2a. The sample flow carries tons of magnetic beads into the nano-sieve channel and is shaped by sheath flow from both side channels as depicted in Fig. 2a-i. The deformation of the PDMS roof was induced by a large pressure drop from the applied flow rate, allowing the migration of those magnetic microparticles.³⁵ The applied magnetic beads are predictably confined and gradually accumulated in the boundary of the active pneumatic layer as shown in Fig. 2a-ii, until the well-established bead stacking was observed. Once the beads are stacked within the channel, DI water containing a fluorescent component is pumped into the channel at a constant flow rate of 8 μL min⁻¹, and the applied sheath flow per channel is tuned from 5 to 15 μL min⁻¹ for highlighting the focused stream and testing the minimum “working width” of the focused stream as shown in Fig. 2b. The minimum “working width” was found and measured at a flow rate ratio of 3.75 : 1 (sheath flow/sample flow), corresponding to 15 μL min⁻¹versus 8 μL min⁻¹, as described in Fig. 2c, which states an excellent linear relationship. As the experiment progressed, we did not observe that the number of stacked beads significantly changed, therefore further confirming the stability of this nano-sieve system.

Fig. 2 (a) The bright-field pictures showing the profile of the focusing stream caused by the sheath flow (a-i) and the formation of bead stacking in the channel (a-ii). (b) The visualization of the focused mainstream displaying the relationship between the “working width” and flow conditions. The inset picture shows the sketch of the designed nano-sieve channel. (c) The linearity of the measured width as a function of flow ratio for both theoretical calculation and experiments.

Characterization of the flow ratio regarding the concentration effect

To explore the effect of sheath flow/sample flow ratio on target concentration, three different ratios were defined as low ratio, middle ratio, and high ratio for testing, corresponding to the measured “working width” of 1785.46 ± 8.69 μm, 1179.92 ± 9.47 μm, and 808.40 ± 2.51 μm, respectively, as shown in Fig. 3a-i–iii. The relative width of the sample flow, w_m/w, is compared between experiments and the equations of eqn (1) through eqn (3) in Fig. 3b. The results show that the simple flow rate theory can well simulate experimental results, and the assumptions of constant channel height are valid. In addition, this good fitting also indicates that the laminar flow in the microfluidics has limited mixing before the barrier.

Fig. 3 The concentration effect under various sheath flow/main flow conditions. (a) The fluorescence images visualize the measured “working width” related to the various flow conditions of low ratio (a-i), middle ratio (a-ii), and high ratio (a-iii). (b) The result comparison between the theoretical model and experiments. (c) The quantitative data present the concentration effect under different flow conditions. (d) The initial sample solution of fluorescent nanoparticles (1.71 × 10⁵ beads per mL) detected on a glass slide. (e) The concentrated nanoparticles were observed at (e-i) 4-fold magnification and (e-ii) 20-fold magnification, respectively.

In each experiment, 150 μL of green fluorescent bead solution (1.71 × 10⁵ beads per mL) was injected into the bead stacked nano-sieve channel under a constant flow rate of 8 μL min⁻¹ and the related sheath flow rate per channel of 5, 10, and 15 μL min⁻¹, respectively. The data of the measured integrated density from each captured fluorescence image are plotted in Fig. 3c. In a total recording time of 16 min, the fluorescence signal was significantly increased under each tested condition, indicating the accumulation of target particles within the proposed nano-sieve system. When a high ratio of sheath flow (15 μL min⁻¹)/sample flow (8 μL min⁻¹) was employed, the fluorescence signal at 16 min was largely enhanced compared to the other two conditions, which shows ∼8.7-fold enhancement related to the fluorescence signal under the condition of a low ratio. Then, we applied the physical model related to equations eqn (4) through eqn (8), to fit the experimental data to obtain the key parameters in the model to gain insights into the process. The fitting results are shown in Fig. 3c, which show that the semi-empirical model can capture the major trend of the experiment. The fitted results are listed in Table 1. The result of k₂ = 2.83 indicates that the sinking rate of the beads is sensitive to the transport velocity. The good fitting further indicates that a simple mechanism of bead sinking can be used to explain the major process. At a high sheath flow ratio, the bead stacking can reach saturation more quickly, since the fitting model only considers the impact of transport velocity. In this study, we used high concentration (1.71 × 10⁵ beads per mL) of nanoparticles. However, the goal of this work is to efficiently concentrate and sensitively detect very low-concentration bacteria from applied samples. Therefore, the ratio of the bacteria to the magnetic stacked beads is very low without saturation problems. The good comparison at the high sheath flow ratio indicates that velocity is the major factor of the process. More complicated processes might be present in the medium and low flow ratios, in which bead stacking may depend on flow and stacking uniformity, stacking compression, etc.

Table 1 The parameters of the fitting result

F 0	k 1	k 2
5.55 × 10^5	0.176	2.83

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The initial fluorescence bead solution placed on a glass slide can be barely recognized under the fluorescence microscope with 20-fold magnification, as shown in Fig. 3d. From Fig. 3e, however, at the middle sheath flow ratio, the efficient concentration of those fluorescence beads can be successfully achieved. The fluorescence signal can be easily detected under the fluorescence microscope at low magnification of 4-fold (Fig. 3e-i). The enhanced fluorescence signal can be realized in great detail, by using the higher magnification of 20-fold (Fig. 3e-ii).

Accumulation of applied particles in the confined region

To further evaluate the reliability of this nano-sieve system for consistently concentrating the target particles, 250 μL of green fluorescent nanoparticle solution (1.71 × 10⁵ beads per mL) was introduced into the bead stacked nano-sieve channel (Fig. 4a) at the high ratios of 15 μL min⁻¹ (sheath flow) and 8 μL min⁻¹ (sample flow) through a total time of 30 min. The fluorescence signal was monitored, and the fluorescence images (Fig. 4b-i–vi) were captured every 5 min for further analysis. It is noticed that the trapped fluorescent nanoparticles are not uniformly distributed across the nano-sieve channel, especially for a central region where there appears to accumulate less nanoparticles. This phenomenon may be caused by the initial movement of the nanoparticles that are randomly released from the sample inlet, and by the secondary migration after sheath flow injections. Then, more nanoparticles are potentially moving to one side of the mainstream in a lateral direction.³⁶ However, it could not critically impact the capacity of this nano-sieve system, which can be quantitatively validated by the increased fluorescent signal in a specifically confined region with the elapsed time. The plotted data from the measured integrated density of each captured image present that the fluorescence signal across the channel is gradually increased, demonstrating the reliable accumulation of target particles, even under the high ratio condition (Fig. 4c).

Fig. 4 The fluorescence signal monitoring for accumulated fluorescent nanoparticles (green) at the specific region. (a) Bead stacking within the nano-sieve channel under the high flow ratio condition. (b) The fluorescence images presenting the concentrated target nanoparticles for the processing times of (b-i) 5 min, (b-ii) 10 min, (b-iii) 15 min, (b-iv) 20 min, (b-v) 25 min, and (b-vi) 30 min. (c) The measured integrated density as a function of elapsed time in a total time of 30 min.

Sheath-enhanced concentrations of MRSA bacteria

After optimizing the best experimental conditions with 0.5 μm fluorescent beads, we then studied on-chip concentration and detection with MRSA bacteria. To validate the effectiveness of the sheath-assisted and pneumatic-regulated nano-sieve, we employed a fluorescence labeling method as a straightforward approach for real-time bacterial imaging and quantification.³⁷ The MRSA sample solution with a concentration from 100 to 10⁴ CFU mL⁻¹ was tested under the high ratio condition, which is related to the flow rate setting of 15 μL min⁻¹ for sheath flow and 8 μL min⁻¹ for sample flow. From the image panel in Fig. 5, below the concentration of ∼10⁴ CFU mL⁻¹, MRSA bacteria on the glass slide are hardly realized under the fluorescence microscope as shown in Fig. 5a-i and ii. The detection limit of the fluorescence microscope was found at ∼10⁴ CFU mL⁻¹ with the sample solution on the glass slide (Fig. 5a-iii). In contrast, the proposed nano-sieve system shows distinct fluorescence signals by concentrating the MRSA sample solution at ∼10⁴ CFU mL⁻¹. The image in Fig. 5b-iii shows several tens of stained MRSA bacteria confined in a specific region, which is caused by the sheath-enhanced concentration approach. The quantitative comparison of the integrated density of the fluorescence signal from the off-chip sample (Fig. 5a-iii) and on-chip sample (Fig. 5b-iii) exhibits an impressive on-chip concentration factor of 31.59 ± 1.88 folds. Still, the 10³ CFU mL⁻¹ MRSA sample can be clearly recognized (Fig. 5b-ii). Remarkably, the extremely low-concentration MRSA (∼100 CFU mL⁻¹) can be efficiently concentrated and detected (Fig. 5b-i), further validating the capability of this nano-sieve system not only for on-chip pathogen detection but also for downstream analysis. It is important to emphasize that fluorescence labeling is not the sole approach for in situ bacterial detection. In future applications, our nano-sieve device could integrate with label-free computer-vision algorithms for pathogen detection.^38–40 Alternatively, extensively documented methods for pathogen identification offer another viable approach.^41,42

Fig. 5 The image panel exhibits the comparison of on-chip and off-chip results under the fluorescence microscope. (a) MRSA bacteria sample solutions with three different MRSA concentrations of (a-i) 100 CFU mL⁻¹, (a-ii) 10³ CFU mL⁻¹, and (a-iii) 10⁴ CFU mL⁻¹, were placed on glass slide. (b) MRSA bacteria sample solutions with three different MRSA concentrations of 100 CFU mL⁻¹ (b-i), 10³ CFU mL⁻¹ (b-ii), and 10⁴ CFU mL⁻¹ (b-iii), were captured within the nano-sieve channel. The stained MRSA bacteria present blue fluorescence. Blue arrows indicate the detected targets. The inset shows the bacteria identification with higher magnification.

Typically, microfluidic systems based on standard flow cytometry provide a high-throughput analysis method that is usually combined with fluorescent antibody-induced immunoassays,⁴³ enabling the sensitive identification of target MRSA bacteria in fluid samples for achieving a detection limit from 10³ to 10⁵ CFU mL⁻¹. In a specific case, the detection limit can be improved to ∼100 CFU mL⁻¹ with the employment of a dual aptamer-based detection system.⁴⁴ Some innovative techniques for detecting MRSA bacteria have been developed in recent years, such as PCR⁴⁵ and ELISA,⁴⁶ resulting in a promising detection limit ranging from 100 to 1000 CFU mL⁻¹. However, these methods require a series of delicate procedures, including target purification, immobilization of antibodies, and accurate temperature control, leading to a longer processing time of several hours. In this study, we are developing our nano-sieve system to rapidly and sensitively detect low concentration MRSA bacteria, reaching a detection limit of ∼100 CFU mL⁻¹, but only in a shorter processing time (∼30 min). The target MRSA stained with DAPI dye can be readily distinguishable in a locally confined and desired region with a fluorescence microscope. This proposed method could significantly pave the way to process single-target detection in clinical applications.

The particle focusing methods assisted by sheath flow in microfluidics, such as sheath-assisted spiral channels^47,48 and a straight channel using sequences of pillars,^49,50 typically requires deliberate and tedious trial-and-error process, increasing the difficulty and complexity of both experimental operations and fabrication procedures. Our sheath-assisted nano-sieve device is based on a straightforward fabrication procedure, including the well-established photolithography and wet etching technique. The stable flow conditions can be simply and directly regulated only by the ratio of the applied flow rate (sheath flow/sample flow), which has been validated by an excellent linearity relationship. In this regard, our current nano-sieve system offers stable and reliable flow conditions for efficiently concentrating and sensitively detecting targets in a desired region. Furthermore, the region of beads stacking is wider compared to the inlet area along the nano-sieve channel. This diverging configuration is beneficial to the flow regulation along the centreline, especially at the interface of stacked beads for effectively concentrating targets and expelling liquids from the sample solution. Consequently, the extremely low-concentration MRSA bacteria can be successfully detected in a specifically defined region within the channel. With a higher 20-fold magnification under a fluorescence microscope, ∼100 CFU mL⁻¹ of MRSA bacteria can be sensitively recognized.

On the other hand, the large-volume sheath fluids are usually needed to align and focus the target particles within the flow chamber, which is necessary for them to be detected in a small region while diluting the original sample.²⁶ The well-established bead stacking within the nano-sieve channel, including a mixture of 10 μm and 1 μm magnetic beads, could enable the compensation for the original sample dilution from sheath fluids. The trend of the sheath/sample flow rate ratio demonstrates the accumulation of target nanoparticles in a specific region regulated by sheath fluids, from three different experimental settings. The higher ratios of 15 μL min⁻¹ (sheath flow) and 8 μL min⁻¹ (sample flow) causing the narrower width of focusing flow result in a smaller region but containing more target nanoparticles, presenting the enhanced fluorescence signal from the designed experiment. In this regard, the sheath/sample flow rate ratio could be further theoretically and experimentally studied for reaching the desired concentration of target particles with this nano-sieve system,²² which may pave the way for rapidly, efficiently, and sensitively detecting infectious pathogens from samples at extremely low concentrations below 100 CFU mL⁻¹.

Additionally, in this study, our nano-sieve system is coupled with a pneumatic-induced layer to better counterbalance the hydrodynamic pressure within the channel,^29,51 significantly increasing the reliability of this system for detecting the targets in a local confinement. This unique characteristic is beneficial to the special design of a nano-sieve channel only with a very low aspect ratio of 1 : 20 000 in depth and width, leading to an extraordinary reduction in hydrodynamic pressure within the channel, which is further beneficial to the stability of 3D microbead stacking. In other words, the lack of the robust pneumatic layer and the specifically designed channel can result in the leaking of those microbeads established within the channel, under some flow conditions.³² On the other hand, the formation of magnetic bead stacking could be induced and guided by following the external magnetic field, which could be promising to design a specific assembly of a 3D filter as expected, further optimizing the configuration of stacked beads and enhancing the device capability for efficiently trapping and concentrating the target bacteria with lower concentration below 100 CFU mL⁻¹. More importantly, leveraging this flexible pneumatic chamber under a negative pressure, the captured MRSA bacteria and magnetic bead stacking can be easily released from the nano-sieve channel by the washing process with only a few microliters of fresh PBS solution. And the target bacteria can be efficiently separated from the collection by using an external magnet, which would be directly applied for downstream genotype analysis, such as PCR.

Hence, as already discussed, the proposed approach by combining the flow focusing technique and 3D stacked beads within a nano-sieve device leads to the sheath-enhanced concentration and on-chip detection of applied nanoparticles and MRSA bacteria in PBS solution. With further optimization of the chip design and flow conditions, this proposed method shows promise for achieving enhanced concentration effects and increased target sensitivity.

Conclusions

In this study, we reported a powerful and reliable nano-sieve device for on-chip concentration and detection of target MRSA bacteria with a low concentration of ∼100 CFU mL⁻¹. By leveraging an optimized 3D bead-stacked microstructure and pneumatic-induced layer, the hydrodynamic flow within the specifically designed diverging channel can be well controlled for efficiently concentrating targets, without sample dilution that is usually caused by general sheath-assisted methods.²² Moreover, the application of sheath-assisted flow is beneficial to the movement of target particles from sample solution, by focusing those particles into a desired confined region. The high flow ratios of 15 μL min⁻¹ (sheath flow) and 8 μL min⁻¹ (sample flow) were determined for use in this case, as they present the best concentration effect for those applied target particles, compared with a low flow ratio. Hence, we achieve an impressive on-chip concentration factor of 31.59 ± 1.88 folds while concentrating MRSA bacteria at 10⁴ CFU mL⁻¹ with our nano-sieve system less than 30 min. In addition, our physical models further rationalized the working principle of such a nano-sieve device, aiming to optimize the current nano-sieve system, such as balancing the dimension of the channel and the flow ratio, to process the single-target on-chip detection for clinical applications.

Data availability

Data for this article are available at the UC-Riverside Google Driver system.

Author contributions

Xinye Chen: conceptualization, methodology, investigation, data curation, formal analysis, writing – original draft, and writing – review and editing; Ruonan Peng: investigation and writing – original draft; Ruo-Qian Wang: methodology, investigation, writing – original draft, and writing – review and editing; Ke Du: conceptualization, methodology, investigation, funding acquisition, supervision, and writing – review and editing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by NIH NIGMS R35GM142763 and The Rural Development Administration of the Republic of Korea (A01008_57000_K014475001).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4lc00698d

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