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Lee, S.M.; Balakrishnan, H.K.; Doeven, E.H.; Yuan, D.; Guijt, R.M. Cell Lysis. Encyclopedia. Available online: https://encyclopedia.pub/entry/51634 (accessed on 19 August 2024).
Lee SM, Balakrishnan HK, Doeven EH, Yuan D, Guijt RM. Cell Lysis. Encyclopedia. Available at: https://encyclopedia.pub/entry/51634. Accessed August 19, 2024.
Lee, Soo Min, Hari Kalathil Balakrishnan, Egan Hendrik Doeven, Dan Yuan, Rosanne Marieke Guijt. "Cell Lysis" Encyclopedia, https://encyclopedia.pub/entry/51634 (accessed August 19, 2024).
Lee, S.M., Balakrishnan, H.K., Doeven, E.H., Yuan, D., & Guijt, R.M. (2023, November 15). Cell Lysis. In Encyclopedia. https://encyclopedia.pub/entry/51634
Lee, Soo Min, et al. "Cell Lysis." Encyclopedia. Web. 15 November, 2023.
Cell Lysis
Edit
This entry is adapted from the peer-reviewed paper 10.3390/bios13110980

Cell lysis is the first step of sample preparation for nucleic acid (NA) detection. Its purpose is to release NAs from the cells by disrupting the structure of cell membranes, which are also known as phospholipid bilayer membranes or plasmalemma. As NA detection typically relies on amplification using polymerase chain reaction (PCR) or isothermal alternatives, carry-over of inhibitory agents including the reagents used for lysis needs to be avoided or minimised. While washing steps are easily implemented in a laboratory setting, for Point of Need testing, the trend is towards substitution of strong inhibitory lytic reagents for more benign alternatives to minimise processing steps and reagent use. Additionally, sustainability of the reagents and their disposal are growing concerns. Here, an overview of lysis methods is provided from the perspective of their suitability for for point of need testing.

nucleic acid amplification testing sample preparation cell lysis

1. Introduction

Cell lysis is the first step of sample preparation. Its purpose is to release target NAs from biological samples by disrupting the structure of cell membranes, which are also known as phospholipid bilayer membranes or plasmalemma. These membranes are part of the cell’s cytoskeleton and control the transport of materials in and out of the cell, as well as communication with other cells [1]. The upcoming section will analyse various lysis methods that employ detergents, enzymes, alkaline reagents, chaotropic reagents, and other reagents. These methods will be evaluated based on their potential to extract NA for PONT application in chemical perspectives. Additionally, the potential microfluidic platforms for PONT application will also be analysed based on miniaturisation capabilities. A non-comprehensive overview of different chemical lysis approaches reported in the literature is provided in Table 1.
Table 1. Chemical lysis approaches with potential for NA-PONT.
Main Lysis Method Secondary Lysis Method Reagents Time/Temp
(min or h/°C)		 Target Sample Matrix Amplification Lysis Efficiency Yield
Recovery Rate
LOD		 Ref.
Detergent BSA 0.3% IGEPAL CA-630
0.1% BSA		 5 min/on ice Mammalian N/A RT-qPCR N/A N/A
N/A
10 cells		 [2]
Ethanol 0.008% Q. saponaria
5% (w/v) NaCl
5% (v/v) ethanol		 48 h/55 °C Yeast N/A N/A 99.0% N/A
N/A
N/A		 [3]
Enzymatic Lysozyme
Proteinase + SDS		 1. 1 h/45 °C
2. 5 h/50 °C		 Soil microbiome Soil N/A N/A ~24 µg/g
N/A
N/A		 [4]
Enzymatic 10 mg/mL Lysozyme
20 ng/mL Proteinase K
0.1% SDS
1 mM EDTA
10 mM Tris-HCl
1 µL RNase		 10 min/N/A Bacteria Milk
(Spiked)		 dRPA N/A ~20 ng/µL from 10 cells
N/A
10 cells		 [5]
Enzymatic 0.5% SDS
1 mg/mL Proteinase K
10 mM Dithiothreitol		 15 min/65 °C Virus Serum
(Spiked)		 RT-RPA N/A N/A
N/A
500 copies/mL		 [6]
Enzymatic 10 mM Tris-HCl (pH8)
10 mM EDTA
1% SDS
10% Triton X-100
Proteinase K
DMS		 20 min/56 °C Virus Clinical RPA N/A N/A
95%
10 copies		 [7]
Alkaline N/A 1.10 mM NaOH
2. 1 mM HCl		 5 min/N/A Mammalian Blood
buccal swabs, saliva, cigarette butts		 qPCR N/A 21.8 ng/µL
N/A
N/A		 [8]
0.5 M NaOH
10 mM Na2EDTA
(pH 8)		 1 min/N/A Plant Plant RT-RPA N/A N/A
N/A
20 copies		 [9]
Surfactant 0.2 M NaOH
1% SDS (dried)		 N/A Bacteria Aerosol
(Spiked)		 qPCR N/A N/A
10%
101 CFU		 [10]
PEG 60% PEG200
20 mM KOH
(pH 13.3–13.5)		 15 min/RT Human
Animal
Bacteria
Plant		 Raw samples PCR N/A N/A
N/A
10 pg		 [11]
1.25% PEG 200
10% PEG 8000
5% (v/v) NaOH		 1. 3 min/RT
2. 10 min/70 °C		 Virus Whole blood
(Spiked)		 LAMP 100% N/A
N/A
102 PFU/mL		 [12]
6% PEG 200
0.08% NaOH		 3 min/RT Fungal Strawberry
(Spiked)		 RPA N/A N/A
N/A
100 fg		 [13]
6% PEG 200
0.08% NaOH		 3 min/RT Oomycete Leaf RPA N/A N/A
N/A
500 fg		 [14]
60% PEG 400
100 mM KOH		 N/A Human Whole blood PCR N/A N/A
N/A
N/A		 [15]
50 g/L PEG 4600
20 mM KOH
(pH 13.5)		 2 min/N/A Fungal
(mycelium)		 Plant LAMP N/A N/A
N/A
19.9 pg/µL		 [16]
Chaotropic N/A 5 M GuHCl 5 min/RT Bacteria Liquid stool
(clinical)		 PCR 50% (G−)
60% (G+)		 Ave 109.5 ng/µL
(3-chamber)
Ave 59.3 ng/µL
(5-chamber)
~60%
N/A		 [17]
4 M GUSCN
20 mM Tris-HCl
1 mM DTT
pH 7.7		 N/A Animal Mixed meat qPCR N/A N/A
N/A
0.1%		 [18]
Enzymatic GuHCl, Proteinase K 30 min/56 °C Bacteria Human saliva PCR N/A 157.2–165 ng/µL
7.86–8.25 µg
N/A		 [19]
Proteinase K
GUSCN		 10 min/56 °C Bacteria Human urine
Milk		 qPCR N/A N/A
N/A
5 CFU/10 mL		 [20]
6 M GuHCl
Proteinase K pH 6.1		 10 min/56 °C Virus Buccal swab
(spiked)		 LAMP N/A N/A
N/A
N/A		 [21]
Detergent 6 M GuHCl
2% Triton X-100
13 mM EDTA
10 mM NaCl
51 mM Tris
(pH 5.5)		 5 min/RT Virus Serum
(Spiked)		 RT-PCR N/A 1.3–2.0 µg/100 µL
N/A
N/A		 [22]
AMP (Melittin, Bombolitin III, MSI-78, or MSI-594) 5 min/RT Bacteria N/A qLAMP 100% N/A
N/A
N/A		 [23]
5 M GuSCN
100 mM EDTA
0.5% (v/v) Sarkosyl		 5–10 min/N/A Bacteria N/A N/A N/A N/A
N/A
N/A		 [24]
6 M GuHCl
2% Triton X-100
13 mM EDTA
10 mM NaCl
51 mM Tris
pH 5.5		 5 min/RT Bacteria Serum
Saliva
(Spiked)		 dRPA N/A 15–35 ng/µL
89.4%, 79.6% (saliva, serum) 1.1 × 108 copies/μL		 [25]
1.5 M GuHCl
50 mM Tris [pH 8]
100 mM NaCl
5 mM EDTA
1% Tween-20		 10 s/RT Fish Blood (Fish) PCR N/A N/A
N/A
104 cells		 [26]
4.8% GuSCN
5% Triton X-100 (pH 6.8)		 N/A Virus Spiked blood PCR N/A N/A
N/A
5 particles		 [27]
4 M GUSCN
1% Triton X-100
1% ß-mercaptoethanol
10 mM 2-Ethanesulfonic acid		 5 min/RT Virus Nasopharyngeal swab LAMP N/A N/A
N/A
1–10 copies/µL		 [28]
4 M GuSCN
10 mM Tris-HCl (pH 8)
1 mM EDTA (pH 8)
0.5% Triton X-100
300 µL Isopropanol
3 µL ß-mercaptoethanol
5.6 µg poly-A carrier RNA
(For RNA)		 3 min/RT Synthetic DNA Synthetic sputum
(Spiked)
Residual urine sample		 qPCR N/A N/A
10.2 ± 4.03%, 91.2 ± 7.46%
(sputum, urine)
N/A		 [29]
0.1 M Tris-HCl (pH8.0)
10 mM EDTA
1% SDS
10% Triton X-100
Proteinase K
DNase I (RNA)		 10 min/RT
(RNA)
20 min/56 °C
(DNA)		 Mammalian
Bacteria		 N/A RT-qPCR
qPCR		 N/A ~100 ng/µL
N/A
103 CUF/mL, 101 cells/mL (DNA, RNA)		 [30]
MIL Peptide
Enzymatic
Detergent		 6 µL [P6,6,6,14+] [Ni(HfAcAc)3−] 1 h/N/A Plant Plant qPCR N/A ~8 ng
N/A
N/A		 [31]

2. Chemical Cell Lysis

2.1. Detergents

Detergents (or surfactants) break down the phospholipid bilayer by virtue of their amphiphilic properties. The membrane solubilisation induced by detergents can be understood in three stages [32][33][34]. Initially, detergent monomers gradually penetrate the outer layer of the membrane, disrupting the orderly arrangement of its molecular architecture. Then, the bilayer becomes saturated with detergent, resulting in phospholipid–detergent mixed micelles. The increasing surfactant content alters permeability and disrupts the osmotic equilibrium of the membrane. This phenomenon forces the detergent-enriched bilayer to fragment and transform into thread-like amphiphilic micelles, leading to complete solubilisation of the bilayer.
Detergents can be ionic and non-ionic, depending on the nature of the polar head. Ionic detergents have charged polar head groups, either positively charged (cationic) or negatively charged (anionic). Cationic detergents often contain ammonium or pyridinium head groups and are used in DNA extraction and cell lysis because the positively charged nature helps disrupt cell membranes and solubilize biomolecules. While anionic detergents, commonly with sulphate or carboxylate ions, are often used in protein electrophoresis, non-ionic detergents have uncharged polar head groups and are suitable for a wide range of applications where ionic interactions should be avoided.
The non-ionic detergent Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl) phenoxy] ethanol) was used for cell lysis in a capillary, mixing a 0.1% (v/v) solution with the sample diffusion owing to the laminar flow regime. Complete lysis of green fluorescence protein (GFP)-expressing cells was achieved within 1 min [35]. In contrast, when Escherichia coli (E. coli) were incubated in 1% Triton X-100 at room temperature for 5 min, only about 10–15% of viability was observed owing to the stronger bacterial walls and the E. coli cell permeability was enhanced to 30% with the aid of additional 1 mg/mL lysozyme [23]. Furthermore, a three-detergent method combining the anionic sodium dodecyl sulphate (SDS), Tween 20, and Triton X-100 (STT) was reported for lysis before RNA extraction from several Gram-negative bacteria, including Pseudomonas putida, Burkholderia cepacia, Agrobacterium tumefaciens, E. coli, and Edwardsiella tarda, and Gram-positive Bacillus subtills [36]. The quantity of RNA extracted using STT buffer was distinctly greater than single-detergent methods with 2 and 5% SDS, according to the gel electrophoresis analysis. Le et al. investigated a lysis solution containing 0.3% of the non-ionic detergent, IGEPAL CA-630, and 0.1% bovine serum albumin (BSA) to lyse circulating tumour cells (CTCs) [2]. The protocol required a 5 min single step on ice prior to direct reverse transcription (RT)-qPCR to detect RNA from CTCs. The IGEPAL CA-630, octylphenoxypolyethoxyethanol, method outperformed a commercial kit when cell counts were 10 and 100; however, at cell counts around 1000, the higher concentration of RNases degraded target RNA and cell debris inhibited amplification, limiting the effectiveness. The result that detergent-induced lysis can be efficiently performed for low-cell-count samples was also agreed with a buffer containing 0.1% Triton X-100 which was used for 1 min lysis of a single cell [35].
The use of detergents in cell lysis has an impact on different biological samples. Detergents like Triton X-100 are widely used in different concentrations, depending on their specific application. For instance, Triton X-100 concentrations can range from 0.1% for capillary cell lysis to 1% for E. coli lysis. In addition, higher concentrations and other surfactants can be used to achieve optimal RNA extraction efficiency in different bacterial species. The concentration of IGEPAL CA-630 varies and has different effects on lysing CTCs based on the cell count. In some cases, a low concentration of 0.1% Triton X-100 is effective for lysing single cells.
Although detergent-based lysis is inexpensive and effective for cell lysis, assays using detergents as sole lysis reagents are barely found in NA-PONT applications as the detergent lysis is often slow. The operational time with detergent lysis is usually longer than 1 h under thermal conditions (45–65 °C). Due to its gentle nature, surfactants are often combined with other lysis approaches such as lytic enzymes and/or thermal lysis. Moreover, its lysis efficiency can vary depending on the sample types and the concentration of the amphipaths. Lysis occurs when the concentration of surfactants is close to their critical micellar concentration (CMC) [37][38], and the concentration of the surfactant can be increased if rapid lysis is desired. Excessive surfactant use, however, may lead to bubble nucleation, which may cause practical challenges, including decreased solvent concentration, interrupted electrical and fluidic conductivity, and changes in hydraulic resistance [39][40]. Surfactants can also inhibit the amplification reaction by damaging amplification enzymes due to their denaturing properties effect [23].

2.2. Enzymatic Lysis

In enzymatic lysis, a biocatalyst is used to cleave and digest chemical bonds in the membranes. Enzymatic lysis is often combined with detergent for hard-to-lyse samples or samples in a complex matrix to improve the lysis efficiency as mentioned above [4][5][23]. During cell lysis, proteinase K promotes proteolysis to digest proteins and protects the NAs from DNase or RNase, but it requires thermal activation at 50–65 °C to optimize its activity [5][41][42][43]. The HIV virus in human serum was lysed using 1 mg/mL proteinase K and 10 mM dithiothereitol (DTT) mixed with 0.5% SDS and used in conjunction with a paper-based isotachophoresis (ITP) device and RT-RPA. The method allowed for the detection to be as low as 500 copies of viral RNA from 1 mL of spiked serum samples [6]. A similar lysis buffer containing 1% SDS, 10% Triton X-100, and proteinase K (concentration not reported) was used to lyse human adenovirus (HAdV). The recovery rate of the viral DNA was 95% with a limit of detection (LOD) of 10 copies of HAdV in the nasopharyngeal samples collected from infected patients [7].
Lysozymes are routinely utilized in NA extraction kits; however, some pathogens (incl. S. aureus) are resistant to lysozyme [44]. Achromopeptidase (ACP), a cocktail of proteases and peptidoglycan-specific hydrolases [45][46], provides an alternative and has been used to lyse Gram-negative bacteria Bordetella pertussis, Gram-positive bacteria Mycobacterium marinum, and S. aureus extensively. As a factor important for PONT, it was also compatible with lyophilisation facilitating storage as a dry reagent. A single, USB-powered platform for bacterial lysis and NA amplification was recently presented using small and large area heaters to deactivate ACP before amplification of DNA specific to methicillin-resistant S. aureus (MRSA), respectively [47]. While ACP required thermal deactivation at 90–98 °C prior to amplification due to its inhibitory effect on polymerases [48] like other lytic enzymes including proteinase K, the thermal degradation step was no longer required owing to the immobilisation of ACP on nitrocellulose paper before enzymatic amplification, simplifying the overall workflow [49]. The lysis efficiency on paper was equivalent to that obtained in test tubes. Although ACPs are reported as the broadly applicable enzymes, the direct comparison with proteinase K and/or lysozymes has not yet been found.
Enzymatic lysis provides effective lysis for hard-to-lyse biological samples and has compatibility with various detergents. Although heat inactivation of lytic enzymes is inevitable for proteinase K before amplification to avoid denaturation of polymerases during the PCR reaction, the enzyme immobilisation technique with ACP made the enzymatic lysis attractive for NA-PONT, with an advantage of enzymatic lysis being that thermal deactivation is no longer required, resulting in a smaller number of sample handling steps.

2.3. Alkaline Lysis

Alkaline lysis (AL) involves the use of high pH to break the fatty acid–glycerol ester bonds in the cell membrane and is often used in combination with a surfactant to aid in the solubilization of the membrane. The first AL protocol was reported in 1979 using a combination of three buffers: Solution I (50 mM glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8.0), Solution II (0.2 N NaOH, 1% (w/v) SDS, pH > 13), and Solution III (5 M potassium acetate, glacial acetic acid, pH 4.8) [50][51][52]. The alkaline conditions as a result of the high concentration of NaOH hydrolyse in the phospholipid membranes and subsequent leakage, fusion, and transformation of the lipid bilayer make the membrane permeable [53]. Following neutralisation with potassium acetate, an ethanol-based precipitation of the DNA allows for its isolation. Though the conventional alkaline lysis method can be time-consuming and pH neutralisation is required before amplification [51], AL has been successfully adapted for PONT applications owing to its effective lysis ability for various sample types.
An automated paper-based microfluidic device utilized AL to facilitate on-chip lysis and DNA extraction from small-quantity (1–2 µL) human blood samples. The blood sample pre-washed with 200 µL of DI water was mixed with 10 mM NaOH (no SDS), and after 5 min incubation, 1 mM HCl was used to neutralize the solution, followed by a washing step of the paper with DI water [8]. The automated protocol yielded about an additional 20–40% of DNA compared with a commercial DNA extraction kit, and it was used for DNA extraction directly from various raw samples, including whole blood, buccal swabs, saliva, and cigarette butts, in a process taking less than 8 min. In addition, the extracted DNA had an adequately high quality for downstream analysis with successful demonstration of STR analysis and DNA sequencing. A rapid pork identification method utilized AL of meat products using 0.2 M of NaOH solution. The meat samples (500 mg) were ground up with 4 mL of the NaOH solution and 5 µL of the resultant extract was mixed with 40 µL of the NaOH solution before thermal incubation at 75 °C for 20 min. The lysate was then neutralized using 360 µL of 40 mM of Tris-HCl (pH 7) and 5 µL of the final resultant solution was used for LAMP amplification. This assay allowed for the detection of 0.5 ng/µL of pork DNA and the 0.1% adulteration of pork in beef mixture [54]. The same AL method was compared with the surfactant cetyltrimethylammonium bromide (CTAB) method, which is a common method for DNA extraction from plant samples, and the result of the RPA–Clustered Regularly Interspaced Short Palindromic (CRISPR)/Cas12a assay showed that the lysis effect of the AL with the aid of a 30 min boiling treatment was comparable with the CTAB method, detecting 0.01% (w/w) pork adulteration [55]. NaOH was used for AL in an assay aiming for the detection of MON863 maize and combined with direct amplification, omitting the extraction and amplification steps. Using a simple 10-fold dilution of the crude cell lysate, MON863 maize was detected after about 8 min of RT-RPA, while the undiluted lysate and its 50-fold dilution attenuated the detection time by 2 min due to inhibition and dilution, respectively [9][56].
AL is faster than lysis using detergent or enzymes. Using AL with 400 mM KOH, 100 mM DTT, and 10 mM ETDA, 80% of E. coli cells were lysed after a 5 min incubation at room temperature, while 1% Triton X-100, 1 mg/mL lysozyme, and their mixture led to only ~30% lysis under same incubation conditions [23]. As speed is important for PONT, this makes AL an attractive option; however, the requirement for neutralisation before amplification may form an operational bottleneck in the development of ideal PONT devices. In the traditional AL method, alcohol precipitation can be considered as another bottleneck due to its process length. In addition, the precipitation is routinely performed with high-speed centrifugation at 4 °C [51], which are unfavourable features for PONT devices, leading to the collaboration of the AL method with SPE approaches.
Chomczynski and Rymaszewski alleviated this neutralisation issue introducing an alkaline polyethylene glycol (PEG)-based (AP) lysis method involving a single step for lysing bacteria, eukaryotic tissue samples, and whole blood, using a single reagent consisting of 60% (w/v) PEG 200 and 20 mM NaOH or KOH (pH 13.3–13.5) [11]. Samples were mixed with 10 times the sample volume of the AP reagent followed by up to 15 min incubation at room temperature. The alkalinity effect of PEG 200 in the presence of a low concentration of KOH rapidly decreased the pH upon dilution with the PCR reaction mix. The AP cell lysate can be subjected to PCR amplification using only a ten-fold dilution in the PCR reagent. The simple workflow of the AP method and its versatile sample range are schematically described in Figure 1B. The AP reagent was modified to 5% (v/v) NaOH, 1.25% PEG 200, and 10% PEG 8000 to detect dengue virus present in whole blood [12]. By using 0.8 g of 50 µm glass beads with rotation for 90 s at 1500 rpm, the lysis efficiency was estimated close to 100% with a LOD of 102 PFU/mL using LAMP. Lu et al. demonstrated the RPA–lateral flow strip assay to detect Phytophthora cactorum in strawberry and P. infestans in potato leaf using a modified AP reagent containing 6% PEG 200 and 0.08% NaOH, and this assay—using a 3 min incubation at room temperature for lysis—was capable of detecting as low as 100 fg and 500 fg of pathogenic DNA, respectively [13][14]. In later work, PEG 200 was replaced with PEG 400 to investigate the alkalinity effect of PEG 400, and optimal lysis was observed when twice the AP volume comprising 60% PEG 400 and 100 mM KOH was mixed with whole blood [15]. Application of the AP method to plant samples was demonstrated using a modified AP buffer containing 50% (w/v) PEG 4600, 20 mM KOH (pH 13.5), and a 10 mm stainless steel bead to improve disruption of the thick cell walls/membrane of the fungus, such as the invasive forest pathogen Heterobasidion irregulare, with the minimum LOD of 19.9 pg/μL by qPCR [16].
AP lysis has streamlined sample preparation for diverse applications, including pathogen detection in plant samples and whole blood. It offers high lysis efficiency and compatibility with various samples and amplification reactions in the absence of neutralisation where pH adjustment can be achieved through dilution with the PCR reaction mix. However, this dilution effect may lead to compromising detection sensitivity. Also, it is essential to note that the alkaline conditions in this method can potentially degrade genomic and plasmid DNA, making careful optimisation of the incubation time necessary [57]. Despite these considerations, the AP method remains a valuable tool for simplifying and expediting sample processing in molecular biology and diagnostic applications.

2.4. Chaotropic Lysis

Chaotropic lysis is based on the disruption of hydrogen bonding, impacting the protein structure, and compromising hydrophobic interactions within the cell membrane [58][59]. Chaotropic agents also denature the NA-degrading nucleases [60], protecting the NAs. Chaotropic reagents yield high efficiency in lysis and NA isolation. The most commonly used chaotropic reagents for cell lysis are guanidium hydrochloride (GuHCl) [19] and guanidinium thiocyanate (GuSCN) [24] in combination with ethanol and they can be readily found in the commercially available NA extraction kits.
Advantages of chaotropic lysis include the fact that it can be performed at room temperature using a short incubation time (e.g., 5 min) and that it can aid in binding the NAs to a stationary phase for NA extraction [17]. However, the appeal of guanidinium salts for lysis is limited by the non-sustainable synthesis, the known inhibition of amplification enzymes requiring additional clean-up, and the hazardous nature that complicates the disposal of PONT devices employing the guanidinium salts, as discussed in more detail below.
Chaotropic agents are also compatible with enzymatic reagents and/or surfactants to enhance the lysis efficiency. For instance, human saliva was incubated at 56 °C for 30 min in a buffer consisting of GuHCl and proteinase K (concentration not reported) followed by an RNase treatment to quantity bacteria, yielding 157.2–165 ng/µL, or a total DNA recovery of 7.86–8.20 µg [19]. A cell lysis buffer containing GuSCN, proteinase K, and ethanol was also reported, employing a 10 min incubation at 56 °C to detect Gram-negative bacteria by qPCR. Using Salmonella enterica serovar Typhimurium, from human urine and fresh milk samples, comparable outcomes to a commercial kit were obtained, reporting a LOD of 5 CFU/10 mL from both sample matrices [20].
The surfactant Triton X-100 is also compatible with chaotropic agent salts for lysis. For instance, 22.92 g GuHCl (equivalent to 6 M), 2% Triton X-100, 0.15 g EDTA, and 0.025 g NaCl dissolved in water giving the final volume of 40 mL facilitated the lysis of Hepatitis B virus (HBV)/Hepatitis C virus (HCV) spiked in human serum samples and yielded 1.3–2.0 µg viral RNA from 100 µL serum spiked with 1000 IU of HBV or HCV via an automated integrated instrument for MB-SPE [22]. The same lysis buffer was adapted into a sample-in-digital-answer-out system to quantitatively detect the pathogenic Mycobacterium tuberculosis (MTB), from human serum and saliva samples. This automated system recovered 89.4% and 79.6% DNA from spiked saliva and serum, respectively, and the assay detection limit was 15 to 35 ng/µL MTB genomic DNA (gDNA) depending on mixing [25]. Tween-20 was combined with 1.5 M GuHCl, 50 mM Tris (pH 8), 100 mM NaCl, and 5 mM EDTA, achieving cell lysis in 10 s at room temperature to detect the targeted viral gene in fish blood. In combination with PCR, a LOD of up to 104 cells was presented, comparable with the performance of a commercial kit [26]. The lysis effect of buffers containing GuHCl and 50 mM Tris (pH 8.0), 0.5% (v/v) Triton X-100, and 1% (v/v) Tween 20 was tested for cucumber mosaic virus (CMV) [61], demonstrating an increase in recovered viral RNA from 105 to 107 RNA copies with increasing GuHCl concentration from 400 mM to 2 M, but the recovery dropped back to 105 when the concentration of GuHCl was increased further to 4 M, suggesting GuHCl-driven inhibition. Interestingly, a recent study reported that a small amount (40 mM) of GuHCl can significantly improve the turnaround time (10 min faster) of colorimetric LAMP for the detection of SARS-CoV-2 [62]. However, the increased amplification time using 80 mM suggests that GuHCl can only be used in low amounts without washing.
GuSCN was also used with Triton X-100 for combined lysis and extraction in a solution containing 4.8% GuSCN, 5% Triton X-100 in 50 mM HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, buffer (pH 6.8), and 2 mg of MBs [27]. Combined with qPCR, the LOD was 5 HBV viral particles in 50 µL whole blood. Using slightly lower concentrations of 1% Triton X-100 and 4 M GUSCN and other reagents including 10 mM 2-ethanesulfonic acid (MES) and 1% ß-mercaptoethanol, the lysis of SARS-CoV-2 virus from clinical nasopharyngeal swabs was realized under vigorous orbital shaking at 900 rpm for 5 min. This protocol allowed for the detection of 10 RNA copies/µL, comparable with a commercial kit [28].
Although the chaotropic agent-based lysis buffers have been broadly applied to lyse samples in complex matrices due to their multiple functions in cell lysis and NA extraction, the high concentration of chaotropic reagents imposes a risk of attenuated amplification, and processing steps are needed to remove reagent residues. Additionally, the environmental aspects in synthesis and disposal decrease the appeal for PONT use.

2.5. New Reagents for Cell Lysis

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic molecules that can permeabilize cell membranes of a broad range of microbes, and hence they are promising lytic agents. A study of the use of AMPs for the lysis of hard-to-lyse bacteria, S. typhimurium and S. aureus, systematically correlated wall structure and AMP activity [63]. AMPs including melittin, magainin analogues (MSI), bombolitin, and cecropin were utilized for lysing bacteria cells in urine samples prior to LAMP [23]. As is shown in Figure 1 of ref. [23], the viability of E. coli reached 0% after the addition of 50 µM of different AMPs (cecopin P1, SB-37, MSI-78, and MSI-594) and 5 min of incubation at room temperature, while no lysis of E. coli was found with melittin or bombolitin III under this condition. However, most AMPs tested severely inhibited amplification by LAMP, except for the cecropins (P1 and SB-37). When performing LAMP directly from crude bacteria lysate with cecropin P1 treatment, the time to positive improved six times compared to untreated or heat-treated samples.
Ionic liquids (ILs) have unique solvating properties and have also been used for lysis of white blood cells and used for the extraction of NAs without significant interference with amplification. ILs are salts with a melting point < 100 °C and hence are liquid at room temperature. Magnetic ILs (MILs) are a subclass of ILs that include a paramagnetic ion [64]. The hydrophobic MIL trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate (II) ([P6,6,6,14+] [Ni(hfacac)3−]) and IL ([P6,6,6,14+] [NTf2−]) were used to lyse different plant species (e.g., Arabidopsis thaliana and Nicotiana benthaminana) within 30–60 s without an additional lysing reagent or heating. Owing to their solvating properties, the NAs were extracted into the MIL with the loaded MIL retained with the help of a magnet allowing for removal of the sample matrix and introduction of the amplification reagents. The MIL facilitated the extraction of 0.5–4 µg DNA from 0.5 mg plant tissue, more than the maximum of 0.6 µg when using [P6,6,6,14+] [NTf2−] [31]. The MIL was compatible with the amplification, attenuating amplification by only 7.9%.
Emerging lysis methods using AMPs and ILs have offered remarkably rapid lysis processes (30 s–5 min), making them attractive for PONT. However, AMP methods have only been used with bacteria (E. coli) which are typically fast to lyse; hence, further testing on more sample types is desirable. ILs also allow for fast lysis and provide a greener alternative to many organic solvents, but the high viscosity [65] and cost of ILs [66] mean that further research is required to enhance their appeal for PONT.

3. Other Cell Lysis Methods

In the early development of microfluidics NA-PONT systems, the focus was to demonstrate amplification and detection, with sample preparation mostly conducted off-chip using commercial kits or instruments. With time, sample preparation protocols have been purpose-developed for PONT use and combined and integrated with NA-PONT systems. Because most of the chemical lysis methods discussed above come with the risk that carryover reagents attenuate amplification, reagent-free approaches including mechanical and thermal lysis methods provide an attractive alternative. A brief overview these chemical-free lysis approaches applied for NA-PONT is provided below.
For example, a stand-alone miniature and battery-operated bead beater, the Omnilyse, was demonstrated to provide similar performance lysing bacteria cells to the benchtop benchmark Biospec Mini Beadbeater [67]. The instrument remains commercially available more than a decade after its introduction, and it has been used in conjunction with PONT testing, including for the lysis of Mycobacterium tuberculosis in sputum [68] and Chlamydia trachomatis in vaginal swabs [69]. An overview of reports on acoustic, piezoelectric, thermal, and electrical lysis relevant to NA-PONT is provided below, with more detailed reviews on mechanical cell lysis methods published elsewhere [70][71].
Acoustic forces can be used for lysis, as the interaction of the sound waves with a liquid medium induces rapid streaming flows that can impart shear stresses on the suspended particulate matter including cells, to the point at which the cell membrane is disrupted. Acoustic lysis is effective for mammalian cells but can be more challenging for bacteria, despite an early report in 2005 demonstrating lysis of B. subtilis spores with 50% efficiency following 30 s of sonication with a 2.5 μL volume [72]. Its potential for PONT was demonstrated by a comparison between a sonication probe in a cup and channel with the Bulk Acoustic Wave (BAW)-based lysis of E. coli demonstrating 50% lysis in 20 s, using 365 times less energy for the channel than for the cup-based approach [73]. Similarly, the use of a traveling Surface Acoustic Wave (SAW) only resulted in an E. coli lysis efficiency of 20% of that of surfactant-based lysis [74]. Cavitation microstreaming employs an acoustic field to vibrate an air bubble trapped in a liquid medium, creating frictional forces at the air–liquid interface that generate a circulatory bulk flow that is experimentally relatively simple to apply. Kaba et al. used cavitation microstreaming for lysis of mammalian cell lines in a purpose-designed microchamber with cavities by attaching a piezoelectric transducer to the microfluidic device, using MBs to bind the freed NAs [75]. Under un-optimized conditions, the performance of the device was just under that of commercially sourced kits; however, this was conducted in half the time with less handling and a dynamic range covering five orders of magnitude. Based on theoretical considerations and simulations [76], Zupanc et al. demonstrated hydrodynamic cavitation on the inactivation of bacteriophage phi6 using cavitation [77] with good integrity of the viral RNA. While showing some potential, these results were obtained at the mm scale with high flow rates aiming for disinfection rather than PONT.
The piezoelectric actuation of micropatterned silicon impactor chips in PDMS devices was used to perform cell lysis by physically breaking microbial cell walls via micromechanical impaction. Despite demonstrated efficacy for mammalian cells, more robust and smaller pathogens typically targeted in PONT are more challenging to lyse using this approach. Different silicon microarray geometries and fabrication technique approaches were compared for the efficacy of lysing two yeast species (S. cerevisiae and C. albicans) to evaluate their efficacy [78]. Despite the effective crushing of beads, the lysis efficiency was estimated < 10% for both species, with future work planned for the optimisation of flow and actuation rates.
As heating is typically required for most NA amplification approaches, thermal lysis appears as an attractive approach. Indeed, a PDMS device was integrated with a carbon paste pad for resistive heating and used for the lysis of Gram-negative Pseudomonas aeruginosa and Gram-positive B. megaterium; however, the lysis efficiency was not quantified [79]. An attractive tube-based method was reported, showcasing the lysis of M. tuberculosis and amplification by helicase-dependent amplification in a single heat incubation step at 65 °C for 60 min; the lysis efficiency was similar to chemical lysis, as quantified through culturing plates [80].
During electrical lysis, the cell membrane is opened by exposing it to a high electric field, leading to the release of the intracellular components. Like other physical lysis methods, the appeal of electric lysis for NA-PONT includes the simple operational setting and no need for reagents. However, for small cells such as bacteria (approximately 1 μm long and 0.5 μm thick), the required electric field to achieve the necessary transmembrane potential for lysis (∼1.5 V) is extremely high (>15 kV/cm), requiring, for example, pulsing regimes to allow for heat dissipation [81]. Electroporation of bacterial cell walls was achieved by applying a low-frequency alternating current (AC) field across interdigitated electrodes, demonstrating highly effective bacterial lysis at 0.5 μL/min, with the efficiency dropping at higher flow rates [82]. Mycobacterium smegmatis was captured onto a packed bed of microscale silica beads and lysed under an ultrahigh intensity (up to 8000 V/cm) [83]. Using electric pulses, lysis was quantitatively assessed using the mRNA copy number per cell for four representative mRNAs in the cell lysate, with the optimum obtained for 30 pulses in 3 min. Overall, electrolysis provided a significantly more complete release of intracellular mRNAs than bead beating, releasing up to 18 times more RNA molecules. Based on the yield dropping off for higher voltages, the authors concluded that the lysis was near quantitative, but the efficiency was not calculated.
Electrical lysis was combined with electrophoretic concentration of bacteria on a nanoporous membrane, using the high potential drop across the membrane for lysis of the concentrated bacteria [84]. The efficiency of the device was determined through bacterial culture of the lysate and was found to be 90% when a potential of 300 V was applied for 3 min. While qPCR was conducted to confirm the quality of the DNA from the lysed cells, further work preventing loss due to non-specific binding and methods to collect the DNA from the lysate are required for interfacing this approach to NA-PONT.
The high field strength demand for electrical lysis was mitigated by combining electrical lysis with mechanical lysis, using ion concentration polarization (ICP) near ion-selective membranes (ISMs) for the formation of fast electro-convective vortices concentrating agitated bacterial cells toward the high field region near the ISM walls [81]. A low electric field (100–300 V/cm) enabled bacterial lysis even in physiological buffer (e.g., 150 mM). While the high (>88%) protein yield demonstrated efficient lysis, the mRNA recovery was only 5%, but it was still better than that obtained using control experiments using bead beating.

Despite encouraging results in the lysis of mammalian cells, non-chemical microfluidic lysis approaches including acoustic, piezoelectric, thermal, and electrical lysis are not as effective in the lysis of smaller and tougher microbial targets as chemical approaches. Consequently, chemical lysis has been most popular for NAAT, and a review of advances in lysis using detergents, enzymes, alkalinity, and chaotropic reagents indicates that combinations of multiple lytic agents often lead to increased lysis efficiency. The quest for reagents with decreased potential for attenuating the amplification reaction has led to viable alternatives to chaotropic agents. For example, AP lysis has enabled direct amplification from the lysate, minimising post-processing to a simple dilution to reduce the alkalinity. Solid-phase extraction, where NAs are bound to a solid support before elution into a purified aliquot for amplification, are popular, because an elution volume smaller than the sample volume can aid in concentration enhancement. An increasing differentiation from standardized approaches developed for laboratory-based NAAT can be seen with the growing number of approaches developed for PONT. In lysis, chemical approaches based on alkaline conditions or detergent/enzymatic approaches are expected to dominate because of the appeal of speed, efficiency, and the little environmental concern; new tailored mechanical lysis approaches are anticipated to be capable of quickly and effectively lysing microbial cells.

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Subjects: Biochemistry & Molecular Biology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Soo Min Lee
,
Hari Kalathil Balakrishnan
,
Egan Hendrik Doeven
,
Dan Yuan
,
Rosanne Marieke Guijt
View Times: 895
Revisions: 2 times (View History)
Update Date: 16 Nov 2023
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