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Feb 26, 2014 - A fast and sensitive assay of protease activity on a micro/nanofluidics preconcentrator combining with fluorescence resonance energy tr...
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Sensitive Assay of Protease Activity on a Micro/Nanofluidics Preconcentrator Fused with the Fluorescence Resonance Energy Transfer Detection Technique Chen Wang,†,‡ Jun Ouyang,† Yun-Yi Wang,† De-Kai Ye,† and Xing-Hua Xia*,† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China ‡ Department of Physical Chemistry, School of Basic Science, China Pharmaceutical University, Nanjing, 211198, China S Supporting Information *

ABSTRACT: A fast and sensitive assay of protease activity on a micro/ nanofluidics preconcentrator combining with fluorescence resonance energy transfer (FRET) detection technique has been developed in a homogeneous real-time format. First, the functionalized nanoprobes are formed by loading dye labeled protein onto gold nanoparticles (AuNPs), in which, the photoluminescence of donor dye was strongly quenched by AuNPs due to FRET mechanisms. For protease activity assay, the nanoprobes are enriched by a micro/nanofluidics preconcentrator. When the target protease is transported to the enriched nanoprobes, cleavage of protein occurs as a consequence of molecular recognition of enzyme to substrate. The release of cleavage fragments from AuNPs nanoprobes leads to the enhancement of fluorescence and enables the protease activity assay on the micro/ nanofluidics chip. As a demonstration, digestion of fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) by trypsin was used as a model reaction. Because of the highly efficient preconcentration and space confinement effect, significantly increased protein cleavage rate and protease assay sensitivity can be achieved with enhanced enzyme activity. The present micro/nanofluidics platform fused with the FRET detection technique is promising for fast and sensitive bioanalysis such as immunoassay, DNA hybridization, drug discovery, and clinical diagnosis.

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protease. The proposed AuNP-based assay method allows a simple visual monitoring of the protease activity in a rapid and efficient fashion. Xia et al.5 demonstrated a gold nanocage and dye conjugate for fluorescence detection of protease activity. By tuning the localized surface plasmon resonance peak of the Au nanocage away from the emission peak of the dye, the protease activity could be detected with relatively high sensitivity. Liu et al.6a added a macroporous ordered silica foam into the conventional in-solution protease reaction system, a fast proteolysis occurred due to the enrichment of both proteases and proteins into the macropores of the reactor. In addition to the approaches described above, another alternative to improve the protease assay is to utilize microfluidics device as the enzyme microreactor due to their obvious advantages such as reduced sample/reagent consumption, high performance and speed, portability, and high degree of integration.9 Liu’s group has successfully developed various microchip-based enzyme microreactors for protease assay including nanozeolites,10 silica sol−gel,11 and layer-bylayer.12 As compared with conventional protease assay in

rotease is an important class of enzymes in the body’s metabolic processes, which is closely related to a large number of vital processes including the cell growth, cell death, tissue remodeling, and immune defense. Since many proteases express at low abundance in biological systems, it is of great significance in life science research to develop highly sensitive approaches for exploring protease activity.1 Up until now, many protease activity assays have been proposed.2,3 Traditional protease activity is monitored in solution consisting of a mixture of the protease and protein(s) being digested, which usually suffers from disadvantages of low sensitivity, longer incubation time, and large sample consumption.2 However, many proteases express at low abundance naturally in biological systems, so there continues to be a high demand for a sensitive and fast assay of protease activity toward the low-level in limited sample quantities. Several strategies have been proposed to improve sensitivity and efficiency of the protease assay. For example, different nanomaterials including nanoparticles,4 nanocage,5 mesoporous materials,6 photonic crystals, 7 and electrospun structures8 have been used for protease activity assay. Lee et al.4a developed a near-infrared-fluorescence-quenched AuNP probe for protease activity determination. When the target proteases met functionalized AuNP probes, cleavage of the substrate occurred and strong near-infrared-fluorescence signals recovered against © 2014 American Chemical Society

Received: January 16, 2014 Accepted: February 26, 2014 Published: February 26, 2014 3216

dx.doi.org/10.1021/ac500196s | Anal. Chem. 2014, 86, 3216−3221

Analytical Chemistry

Article

solution, microfluidics technology enables the implementation of miniaturized space confinement, contributing to the improvement of enzyme reaction rate and efficiency in a shortened incubation time. However, the miniaturized architectures of microfluidics put forward new and specific challenges. For example, flows are laminar in microfluidics and mixing is mainly achieved by the slow diffusion process. Also, the dropped sample amount down to a femtoliter to nanoliter decreases the detection sensitivity. It becomes especially challenging when the sample concentration is at low-level. To overcome these limitations, different nanostructures including nanoporous materials,13 nanoporous networks,14 array nanochannels,15 and a single nanochannel16 have been incorporated into microfluidics devices using as preconcentrator. Because of the charge and size effect of nanochannels, the formed micro/ nanofluidics system has extended functionalities including perfect sample preconcentration capacity,13 accelerated mixing rate,17 and a molecular gates effect,18 which have been successfully employed to efficiently concentrate biomolecules,19 increase the sensitivity of enzyme activity assays,20 fast protein immunoassays,21 and effective protein labeling and following purification on-chip.22 Herein, we propose an assay on a micro/nanofluidic preconcentration device combining with the fluorescence resonance energy transfer (FRET) detection technique to study the protease activity using trypsin as model protease. The micro/nanofluidic device was fabricated using Nafion membrane as the nanojunction connecting two V-shaped microchannels (Figure 1A). The nanoprobes are formed by immobilization of fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) on gold nanoparticles (AuNPs). The fluorescence of FITC will be strongly quenched due to FRET effect between dye and AuNPs. Upon application of an electric field, the nanoprobes can be efficiently preconcentrated

near the nanochannels of the Nafion membrane. When target protease (trypsin) is transported to the preconcentrated nanoprobes, cleavage of protein takes place as the result of special substrate molecular recognition. The release of cleavage fragments from nanoprobes surface leads to the enhancement of fluorescence (Figure 1B) and thus enables the protease activity assay in a homogeneous real-time format (Figure 1C,D). The proposed micro/nanofluidics preconcentration chip combining with FRET detection has effective sample preconcentration capacity and low background interference, allowing a fast and sensitive protease activity assay.

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EXPERIMENTAL SECTION Materials and Reagents. Phosphate buffer of pH 7.0 (10 mM) solution was used as the buffer system. Fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) was obtained from Sigma and used as received. Sodium citrate and HAuCl4 were from Nanjing Chemical Reagent Company. PDMS precursor and curing agent were from Sylgard 184, Dow Corning, Midland, MI. Chemicals and solvents were of analytical purity and were used as received. All aqueous solutions were prepared from deionized water (18 MΩ cm, PURELAB Classic, PALL) and were kept in a freezer to prevent deterioration. All liquid samples were filtered with a 0.22 μm syringe filter to remove particulates before use. Instrumentation. The fluorescence microscopic investigation was performed using an inverted fluorescence microscope (Leica, Dmire2, Germany) equipped with a highly sensitive CCD color video camera (S45, Canon, Japan). NIS-elements BR 2.30 software (Nikon) was used for camera control and image processing. The electric fields to the microchannels were supplied by a laboratory-made high voltage power supply (0− 5000 V) through platinum electrodes placed in reservoirs. The applied voltage can be automatically controlled by a personal computer via an AD/DA converter, and the current is monitored in real time and the corresponding data can be saved in text files. An UV−vis spectrophotometer (Shimadzu UV-3600) was used to measure adsorption of the AuNP solution. Fluorescence spectrometry was conducted using a fluorescence spectrophotometer (Cary Eclipse) with excitation at 480 nm and emission at 518 nm wavelengths. The morphology of spherical gold nanoparticles was observed by high-resolution transmission electron micrograph (TEM, JEOL-JEM-200CX microscope, Japan) operated at 200 kV. A scanning electron microscope (SEM, Sirion, FEI Company, Holland) was used to characterize the thickness of the Nafion between the PDMS/glass gap. Synthesis of AuNPs. Citrate-stabilized gold nanoparticles (AuNPs) were synthesized through thermal reduction of HAuCl4 by sodium citrate following the previous method.23 Briefly, 15 mL of 38.8 mM sodium citrate was immediately mixed with 150 mL of 1.0 mM HAuCl4 refluxing solution under rapid stirring, and then the reactant was kept boiling for 15 min, during which the color changed to deep red. The solution was cooled to room temperature under continuous stirring and then filtered through a 0.22 μm membrane. The sample was centrifuged at 3000 rpm for 18 min and stored at 4 °C before use. The TEM image showed the diameter of such Au NPs as ∼13 nm, and the UV−vis spectrum exhibited a characteristic plasmon absorption band with a maximum at 519 nm (Figure S1A,B in the Supporting Information).The maximum absorbance value is 0.4775, and therefore the AuNPs concentration was approximate calculated as 17.6 nM according to Beer’s law

Figure 1. Strategy for protease activity assay using FRET detection technique combining with the micro/nanofluidic preconcentration chip. (A) Schematic illustration of the micro/nanofluidics chip: (1) trypsin reservoir, (2) AuNPs nanoprobes reservoir, (3−5) buffer reservoirs. The Nafion membrane lies in the center of two V-shaped microchannels as a nanojunction. (B) AuNPs nanoprobes and FRET based protease assays. (C) Protein cleavage on the micro/nanofluidics chip. (D) Monitoring of the fluorescence changes during protein cleavage in real-time. 3217

dx.doi.org/10.1021/ac500196s | Anal. Chem. 2014, 86, 3216−3221

Analytical Chemistry

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Figure 2. (A) Time sequence photo images (a−f) of 1.0 μg mL−1 FITC-DSA in 10 mM PBS (pH 7.0) in the micro/nanofluidics device. Images were taken after applying a voltage of 200 V between the proteins reservoir and waste reservoir for 100, 105, 110, 115, 125, and 130 s, respectively. (B) Plot of 1.0 μg mL−1 FITC-DSA concentration at voltage of 200 V as a function of sample concentration time. The fluorescence intensity is the mean value of the whole fluorescent plug.

using an extinction coefficient of ∼2.7 × 108 M−1 cm−1 at 519 nm for AuNPs according to the previous report,24 which is well in accordance with the reported value.23 Fabrication of AuNPs Nanoprobes. The AuNPs nanoprobes were formed by mixing AuNPs and FITC-DSA solution, incubating for 5 min at room temperature before use.25 The ratio of protein to AuNPs of the nanoprobes was optimized as the following: first, different volumes of AuNPs were added into 100 μL of FITC-DSA solution (10 μg/mL in 10 mM PBS buffer, pH 7.0), then they were diluted by PBS buffer to obtain a final volume of 400 μL, having a final FITC-DSA concentration of 2.5 μg/mL, while keepingAuNPs solutions concentrations, respectively, 0.9, 1.35, 1.8, 2.25, 2.7, 3.15, and 3.6 nM. After completing the mixing, the changes in fluorescence intensity of the reaction mixture versus AuNPs concentrations were recorded for determination of the optimum adsorption ratio of protein to AuNPs. Micro/Nanofluidics Chip Fabrication. In the present work, ion-selective Nafion membrane was integrated in the PDMS/glass gap, lying in the center of two V-shaped microchannels as nanojunctions. The overall layout of the micro/nanofluidic device is represented in Figure 1A. The fabrication of the micro/nanofluidic chip includes three steps: (1) microchannel fabrication on poly(dimethylsiloxane) (PDMS) slab, (2) patterning of Nafion resin on the glass substrate, and (3) bonding of PDMS slab with substrate glass. For fabrication of the microchannel, PDMS base and curing agent were thoroughly mixed in a 10:1 weight ratio and directly casted over an SU-8 photoresist mold on a silicon substrate fabricated by photolithography. The mixture was then heated at 70 °C for 30 min to cure the PDMS precursor. After cooling, the device was peeled off the master, and microchannels on PDMS formed. Before use, the PDMS slab was cut into a proper size and cleaned with methanol and deionized water. For patterning of Nafion resin on the glass substrate, 2 μL of Nafion resin (5 wt %, 663492, Sigma Aldrich) was dropped on the glass substrate. The glass substrate was then placed at room temperature for 10 min for evaporation of the solvent. At last, the PDMS slab with microchannels was bonded with glass substrate through slight squeezing, keeping the Nafion membrane loading in the center of the two V-shaped microchannels, acting as the nanojunction connecting microchannels. In the present work, the height of the microchannel is 40 μm, the width is 150 μm, and the length is 25 mm from reservoirs 1 to 5. Before use, Tween-20 (5% solution in 10 mM PBS, pH7.0) was introduced into the micro/nanofluidic chip

and incubated for several hours. Then, they were completely flushed with PBS buffer solution to prevent nonspecific adsorption of protein onto the microchannels. Protease Activity Assay on Micro/Nanofluidics Chip. First, AuNPs nanopobes were fabricated under the optimum ratio of protein to AuNPs, incubating for 5 min at room temperature. For protease activity assay, 70 μL of nanoprobes was added into reservoir 2 of the micro/nanofluidics chip (Figure 1A), then was concentrated before the Nafion membrane for 5 min by a laboratory-made high voltage power supply. Following that, various concentrations of protease solutions were added into reservoir 1 and electrokinetically driven to meet the preconcentrated AuNPs nanoprobes for protein cleavage reaction. The fluorescent digests released from the surface of AuNPs nanoprobes lead to the enhancement of fluorescence intensity. Fluorescence detection and microscopic investigation were performed using an inverted fluorescence microscope (Leica, Dmire2, Germany) equipped with a highly sensitive CCD color video camera (S45, Canon, Japan). During the course of fluorescence signals recording, the protease solution keeps flowing through the channel. Protease Activity Assay in Bath Solution. To investigate the protease activity in batch solution,the same scheme as that on micro/nanofluidics chip was performed in solution. The fluorescence spectra was measured using a Cary Eclipse fluorescence spectrophotometer with excitation at 480 nm and emission at 518 nm wavelengths. Safety Consideration. The high-voltage power supply should be handled with extreme care to avoid any electric shock.

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RESULTS AND DISCUSSION Characterization of Preconcentration Capacity of the Micro/Nanofluidic Chip. In the present work, Nafion nanoporous material was used to connect microchannels, forming a micro/nanofluidic device (Figure 1A). The flexibility of PDMS and viscosity of Nafion resin enable Nafion membrane to be easily integrated into the PDMS/glass gap in a leak-free manner.26 The Nafion membrane lying between PDMS and glass is ∼800 nm in height (Figure S2 in the Supporting Information).The sample enrichment capacity of the micro/nanofluidic chip was investigated using FITC-DSA as model protein. Upon application of a 200 V voltage to the protein reservoir (anode) and waste reservoir (cathode), the negatively charged FITC-DSA (1.0 μg mL−1) sample is 3218

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Analytical Chemistry

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different ratio of FITC-DSA to AuNPs. The corresponding fluorescence change versus AuNPs concentration is shown in Figure 3B. The results indicate a successful adsorption of FITCDSA onto AuNPs. The optimum ratio of protein to AuNPs (under which the background interference is minimal) is determined as 1.0:14.0 (for the detailed calculation principle, refer to the Supporting Information). Assuming the surface area of FITC-DSA is 25.9 nm2,25 about 68.4% of the maximum monolayer surface density is achieved, which is in accordance with a previous report.25 The submonolayer adsorption density is caused by intermolecular electrostatic repulsion and steric hindrance between neighboring protein molecules absorbed to AuNPs surface. The adsorption of protein to AuNPs is caused by many factors. Under the neutral environment in the present work, free thiols (Cys-34) expose on the surface of globular protein for efficient protein immobilization on AuNPs.25 Furthermore, the -NH2 groups existing in protein can also react with AuNPs. All these interactions together with physical adsorption contribute to the successful formation of AuNPs nanoprobes. Protease Activity Assays. The protease activity was investigated on the micro/nanofluidics preconcentration chip using the FRET analysis technique. The results are shown in Figure 4. It is found that a fluorescence plug in the microchannel appears soon after the introduction of trypsin and then becomes increasingly larger (Figure 4A). Figure 4B shows the corresponding steady-state fluorescence intensity of the reaction product as a function of reaction time for six different trypsin concentrations (from 1 ng/mL to 100 μg/ mL). The inset shows the enlargement of the 1 ng/mL trypsin reaction. The first 50 s is mainly used for sample transport. During this period of time, there is no obvious fluorescence, indicating a low fluorescence background in the present method. Following that, the fluorescence response begins to increase rapidly with the time, indicating a very fast protein digestion process. Then, the fluorescence intensity levels off gradually and finally reaches a plateau. For higher concentrations of trypsin, the time required to get to the plateau is shorter and the change of fluorescence intensity is larger, indicating a faster and more efficient protein cleavage process at higher protease concentration. For the enzyme activity assay, EC50, which is defined as the enzyme concentration at which 50% substrate is converted, is used for analysis. Smaller EC50 indicates a higher enzyme activity. On the basis of Figure 4B, the dependence of the steady-state fluorescence response on trypsin concentration is obtained as shown in Figure 5. As expected, with the increase of trypsin concentration, the fluorescence response shows an almost linear increase from 1.0 ng/mL to 1.0 μg/mL trypsin (shown in the inset of Figure 5), then reaches saturation once trypsin concentration reaches 100 μg/mL. The EC50 value of trypsin is thus evaluated to be 0.514 μg/mL (∼21.6 nM), which lies in the ranges reported previously (from 1 to ∼500 nM).29 The detection limit of trypsin is 0.1 ng/mL (S/N = 3), which is much lower than that of the previous reported assays (0.5 μg/ mL in ref 3 and 0.05 μg/mL in ref 30). For comparison of the trypsin activity in micro/nanofluidics confinement with conventional batch system, the trypsin activity assay was performed under the same conditions in bulk solution. The results are shown in Figure S3 in the Supporting Information. The value of EC50 of trypsin is calculated as 9.35 μg/mL, which is ∼18-fold higher than that on the micro/nanofluidic chip (0.514 μg/mL). The result proves

efficiently preconcentrated near the Nafion membrane, as shown in Figure 2A. The corresponding fluorescence change versus preconcentration time is shown in Figure 2B, which is extracted from Figure 2A using a NIS-elements BR 2.30 software (Nikon). The fluorescence intensity is the mean value of the whole fluorescent plug. Using this device, about 103−104fold protein concentration can be achieved within 220 s. Nafion has a high surface negative charge density in the nanochannels due to many sulfonate groups decorating the hydrophobic polymer backbone.27 Once buffer is filled into the nanochannels, an electrical double layer (EDL) forms immediately at the liquid−solid surface. For 10 mM phosphate buffer (PBS) solution, the EDL thickness is about 3.0 nm.28 If the size ofthe nanochannel is less than 6.0 nm in diameter, the double layeroverlaps in the nanochannel. In the present work, the pore size of Nafion is around 5 nm, with minor changes under different ionic strength conditions.26 Therefore, the double layer overlaps in the Nafion nanochannels and the mass transport depends on the potential in the nanochannels. The co-ions (anions) are excluded from the Nafion nanochannels, while counterions (cations) are drawn into the Nafion nanochannels due to the electrostatic interactions. Therefore, Nafion with highly negative charge density has strong ion selectivity. Once voltage is applied across the membrane, cations can transport through the Nafion nanochannels rapidly from anode to cathode, leading to remarkable depletion of cations in the anode of the Nafion membrane. To maintain the electroneutrality, the anions on the anode side of the Nafion membrane move away from the Nafion membrane. As a result, the ion concentration near the Nafion membrane is reduced, forming the ion-depletion zone (IDZ). Under the two actions of electroosmotic flow (EOF) of the ion-depletion effect, an equilibrium is reached, forming an anion enrichment zone, while cations can pass through the Nafion nanochannels continuously. FITC-DSA is negatively charged under the present experimental conditions and can therefore be efficiently concentrated in front of the Nafion membrane upon application of a voltage. Photos a−f in Figure 2A clearly show the protein enrichment and the ion depletion zone. Fabrication of AuNPs Nanoprobes. AuNPs have good biocompatibility, providing a mild microenvironment similar to that of proteins in native states. In addition, AuNPs can be used as excellent quenchers in the FRET system due to its high fluorescence quenching efficiency and stable optical property. In this work, to fabricate a protease-responsive nanoprobe, AuNPs are used as a matrix to load substrate protein. Figure 3A indicates the fluorescence spectra of nanoprobes with a

Figure 3. (A) Fluorescence emission spectra of FITC-DSA in the presence of various concentrations of AuNPs following incubation for 5 min at room temperature upon excitation at 480 nm. The AuNPs concentration increased from 0 to 3.6 nM (from top to bottom); (B) corresponding fluorescence intensity of the solution as a function of AuNPs concentration. 3219

dx.doi.org/10.1021/ac500196s | Anal. Chem. 2014, 86, 3216−3221

Analytical Chemistry

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Figure 4. (A) Time sequence photo images of the digest products of 10 μg mL−1 trypsin in the micro/nanofluidics device. Images were taken after applying a voltage of 200 V between reservoir 1 and reservoir 4 for 70, 75, 80, 90, 100, 110, 120, and 130 s. (B) Plot of fluorescence response of the reaction products produced with different concentrations of trypsin solutions from 0.001 μg/mL to 100 μg/mL as a function of enrichment time at voltage of 200 V. The inset shows the enlargement of 1 ng/mL trypsin reaction. The fluorescence intensity is the mean value of the whole fluorescent plug.

on the micro/nanofluidic chip. In contrast, in the batch system, there is no obvious fluorescence change within 4 h when trypsin concentration is lower than 100 ng/mL. Therefore, the trypsin assay sensitivity is improved at least by 2 orders of magnitude using the present method. The accelerated reaction rate and assay sensitivity stem from the high preconcentration capacity and space confinement effect of the micro/nanofluidic chip. The generated peptide digests are trapped within the microchannel for further proteolysis to final product, and hence a more efficient protease reaction can be expected. At the same time, the space confinement and high enrichment of substrates increase the encounter probabilities between protease and substrate, leading to a further accelerated reaction rate and reduced reaction time.

Figure 5. Steady-state fluorescence response of the reaction products as a function of trypsin concentrations ranging from 0.001 to 100 μg/ mL.

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that a relative higher trypsin activity can be achieved in micro/ nanofluidics. The enhanced enzyme activity could be due to the confinement of the micro/nanofluidics chip. Our previous studies have shown that a significantly improved enzyme activity appears in micro/nanospace confinement compared to batch systems due to the enhanced protein folding stabilities in the crowded medium.31 To further compare the reaction kinetics on the micro/ nanofluidics chipwith batch system, the time required to reach the final steady-state response in both reaction systems are listed in Table 1. It is found that the reaction time on the micro/nanofluidic chip shortens significantly compared to the batch system. For the same trypsin concentration, the reaction time has been reduced by 30−60-fold on the micro/nanofluidic device. More importantly, the activity of a trace level of trypsin down to 1 ng/mL can be successfully monitored within 10 min

CONCLUSIONS

In summary, a simple micro/nanofluidic preconcentration chip combined with the FRET detection technique has been proposed to assay the homogeneous protease activity. Because of the highly efficient sample preconcentration capacity and space confinement effect of the micro/nanofluidics chip, an accelerated protease reaction rate and higher assay sensitivity can be achieved with an enhanced protease activity. Results show that a trace level of trypsin as low as 1 ng/mL can be successfully assayed on the micro/nanofluidics chip within 10 min. The detection limit of trypsin is as low as 0.1 ng/mL. Compared with the conventional batch reaction system, the assay sensitivity is improved more than 2 orders of magnitude in much shorter time with higher enzyme activity.

Table 1. Reaction Time on Different Devices Ctrypsin (μg/mL) time

batch micro/nano

100

10

1

0.1

0.01

0.001

50 min 100 s

120 min 150 s

200 min 260 s

>240 min 300 s

overnight 480 s