In this section we describe the DEP microbial capture and separation method called Fluid-Screen (FS). First, we provide a brief overview of the design of the FS system followed a detailed verification of the FS method (Section 2.1).
The overall schematic of the operation of the Fluid-Screen System (FS) is shown on Figure 1. In brief, concentration of bacteria in a sample (influent sample contained microbes) is determined by measuring optical density (OD) and by culture and enumerated using PCM to confirm concentration in cfu/mL. The FS system pumps the influent sample through the microfluidic chip with a system of electrodes. The electrodes in the chip generate an electric field. As bacteria enter the chip, electric field captures bacteria on the electrodes. The effluent sample is collected in a tube at the outlet of the chip.
After the Fluid-Screen System processes the entire influent sample and quantifies the number of captured bacteria, effluent sample is collected, cultured, and enumerated using PCM for the confirmation of FS performance.
For details on the engineering design (Section S1.1), microfabrication (Section S1.2) as well as microbial sample preparation (Section S1.3, Section S1.4, Section S1.5) and FS bacterial capture procedure (Section S1.6) see Online Methods.
2.1. Verification of the Fluid-Screen Method
In this section we empirically verify the repeatability of the Fluid-Screen dielectrophoretic capture method (FS). First, we experimentally determine the efficiency of bacterial capture and demonstrate the superiority of the Fluid Screen capture method over the clinically established standard Plate Count Method (PCM) (Section 2.1.1). Secondly, we show that the FS method is equally applicable in capturing very diverse microorganisms, not only bacteria (Section 2.1.2). Lastly, we verify the FS capture method, in a physiologically relevant setting by selective capture of bacterial cells from human red blood cells (Section 2.1.3).
2.1.1. The Efficiency of Bacterial Capture with Fluid Screen System
We demonstrate that the FS system with the Fluid-Screen chip captures 100% of bacteria. Following general guidelines accepted number of colonies for reliable quantification of contamination is between 30 and 25017. For verification of new methods, the US Pharmacopeia (USP) requires that results are within +/-0.5 log. For this reason, the metrics of +/-0.5 log range is the basis for results evaluation with FS method. This paper presents the results on verification of this new method.
Our achieved 100% capture efficiency is much higher than previously reported capture efficiency results, for the similar technology18. We verify the 100% capture efficiency for E. coli-8739 by two different counting methods.
First, we confirm the capture efficiency of the unstained bacteria with the standard Plate Counting Method (PCM), we call it “PCM quantification” (Figure 2a). The capture efficiency is defined accordingly to the following formula:
Second, we visualize captured bacteria in the sample by fluorescence microscopy with SYBR-Green staining and determine their exact number of by a direct “on chip quantification” (Figure 2b). This process demonstrates the real number of bacteria present in the sample.
Figure 2 shows the general schematic of the FS experimental setup for both verification approaches (“PCM quantification” and “on chip quantification”). For the experiment, the influent containing E. coli bacteria was processed on the FS setup. In each experiment 1 mL of the effluent (output sample) was collected and plated immediately on MAC agar plates for enumeration using PCM to calculate the number of Colony Forming Units (cfus). The Electric Field settings allowing for efficient bacteria capture were determined based on a standard in-house calibration protocol (see SI, Sections S1.3 and S1.4).
FS demonstrates an overall 100% bacterial capture efficiency, as verified by “PCM quantification” approach. The unstained E. coli capture experiment was repeated in 3 biological replicates (a biological replicate is new separately grown bacterial sample) with 3 technical replicates (a technical replicate is a triplicate repetition of the FS capture experiment, done sequentially, from the same biological replicate) per each biological replicate for a total of 9 tests (Figure 3 and Figure S9). All 3 biological repeats on FS system, in each of the 9 total conducted experiments, demonstrate 100% bacteria capture efficiency and repeatability (Figure 3 and Figure S9). Detailed data of the “PCM quantification” experiments is summarized in Table S1, including the number of bacterial colonies in the negative control, bacterial concentration in influent, bacterial concentration in effluent and the calculated capture efficiency. The number of cfus in each influent was between 20 cfu/mL and 420 cfu/mL.
Note that in the “PCM quantification” experiment bacteria in influent were not stained with any fluorescent stain. Lack of bacterial staining in a “PCM quantification” experiment avoids any potential growth inhibition by the fluorescent dye on MAC agar plates. For all conducted experiments, acceptable growth, and variability range +/- 0.5 log was reported as recommended by the USP for new method verification. The details on bacterial sample preparation are described in Online Methods, Section S1.3.1.
The direct “on chip quantification” of SYBR-Green stained E. coli is a second approach to experimentally demonstrate the FS 100% bacteria capture efficiency (Figure 3). The direct “on chip quantification” approach is also designed to experimentally demonstrate the superiority of the FS method over “PCM quantification”. The direct FS-counted number of bacteria was determined by background subtraction from total count on the chip (Table 1).
As shown on Figure 3 Fluid-Screen direct “on chip quantification” of captured bacteria is more reliable than a standard established, indirect “PCM quantification”, that requires converting the real number of captured bacteria to cfu/mL values. Most importantly Fluid-Screen direct “on chip quantification” yields a very small bacteria counting error. The small error is a result of a manual operation of the FS system and can be further decreased in future fully automated versions. “PCM quantification” is generally much less reliable, as it is not only indirect, but it introduces multiple human errors (e.g. during sample preparation and dilution, plating on agar plates, etc.). Therefore, standard “PCM quantification” is subjected to a large statistical error that goes beyond the +/- 0.5 log the range accepted by USP.
2.1.2. The Repeatability of Bacterial Capture with Fluid Screen System
We have also assessed the repeatability of the FS capture experiments. The repeatability verification result shows that the FS system demonstrates very high repeatability in the capture and quantification of bacteria. Moreover, the method is more accurate than the required +/- 0.5 log accepted by the USP for new method verification (see Figure 3 and SI Section S2.2; Figure S10, Figure S11, Table S2).
In conclusion, the FS system has demonstrated critical functionality in capturing all bacteria that are present in the test samples, under variable bacterial concentration ranges. The 100% bacterial capture efficiency was verified both by a standard PCM method and by a high-performance direct on-chip quantification. As presented for E. coli the FS system demonstrates very high repeatability of bacterial capture. In addition, it allows to quantify the directly FS-counted number of microorganisms in analyzed samples.
2.1.3 Fluid Screen System Captures Diverse Microorganisms
Fluid-Screen technology can capture and detect very diverse microorganisms. It is not limited to E. coli. It captures both Gram (-) and Gram (+) bacteria, multiple bacterial morphologies, and both individual bacteria and cell aggregates, including bacteria cannot be cultured or do not culture easily (e.g. certain strains of Mycoplasma hyorhinis and Legionella pneumophila). Herein we demonstrate that not only Gram (-) or (+), bacilli or cocci bacteria, but also yeast and molds (including conidia, conidiophores and hyphae) and viruses (data not shown) respond to the electric field, and therefore can be efficiently captured and separated. A total of 40 different species of microorganisms were tested providing the proof of concept for the broad applicability of FS system. All of microorganisms responded to the electric field and were captured, as verified by optical microscopy (Table 2, Table S3). The detailed statistical analysis of the capture efficiency of the other microbial species is going to be presented in future dedicated follow up studies.
2.1.3 Separation of Bacterial Cells from Red Blood Cells
Fluid-Screen system is capable of not only universal capture of diverse microbial organisms, it can also separate them and selectively capture only microbial species of interest. In this section we illustrate the selective capture capability of FS system in physiologically relevant setting, by capturing E. coli bacteria from human red blood cells..
Separation of bacteria from blood is challenging because blood is a complex fluid. Every microliter of blood contains about 5 million red blood cells, in addition to platelets, white blood cells, and proteins. Blood plasma is a high ionic solution containing proteins and ions, which can add to electric screening or chemical non-specific binding, which in turn could lower the efficiency of FS DEP capture. Overcoming such challenges and achieving reliable and efficient detection of bacteria is crucial in clinical diagnostics. For example, to diagnose sepsis, it is required to detect a single bacterium from 1 mL of blood. Efficient and accurate separation and capture of bacteria in blood can result in automated and fast sample preparation on chip.
We have separated E. coli bacteria from human red blood cells. The capture and separation of E. coli from red blood cells was performed using PDMS FS chips (see Online Methods). The results of the E. coli capture from diluted serum in the presence of red blood cells (RBCs), together with the dielectrophoretic conditions of the bacterial capture are summarized on Figure 4. Our results show a E. coli capture and separation from human red blood cells sample. The detailed statistical analysis of the capture efficiency and separation of bacterial cells from physiologically relevant fluids is a domain of future dedicated work.
In conclusion, we have verified the approach and shown that the Fluid-Screen dielectrophoretic method for universal microbial capture is characterized by high efficiency of capture, with no false negatives and false positives. We show that the method is reliable, shows high repeatability, shows fast response and operation. We showed that FS can work on diluted physiological solutions (i.e. human blood) and shows high yield of separation in the presence of blood cells, thus meets the high selectivity requirements. Most importantly, from the clinical perspective, it can process high volumes of liquid to meet clinical testing standards.