Table 2.
Pre-treatment procedures applied to different sample types and in diverse LOC strategies. In details: guanidinium salts, principally GuHCl (Guanidine hydrochloride) and GuSCN (Guanidine thiocyanate), are the most used chaotropic agents with the capacity of solubilizing proteins. Octylphenolethoxylate, commonly known as Triton-X100, is a surfactant used to extract and release the cellular content due to its reactivity with lipid membrane bilayer. DDT (Dithiothreitol) is primarily used to reduce disulphide bonds and, for example, deprotect thiolated DNA (sperm cells) or denature protein structures. Proteinase K is an enzyme used to digest protein contaminants and inactivate nucleases. Tris (tris(hydroxymethyl)aminomethane) is able to maintain pH of the buffer at a stable point, usually 8.0, and to interact with lipopolysaccharides of the membrane, destabilizing it; EDTA (ethylenediaminetetraacetic acid) is a chelating agent which binds to metal ions (e.g., magnesium and calcium), making them unavailable for other reactions such as those related to DNases activity, or breaking Gram-negative bacteria walls. SDS (sodium dodecyl sulphate) is a protein denaturing detergent. CTAB (cetyltrimethylammonium bromide) solubilizes membrane lipids and promotes cell lysis. Lysozyme is an antimicrobial enzyme able to break thick peptidoglycan structures and lyse Gram-positive bacteria, useful when aiming at infection detections. NaOH is used to perform alkaline lysis of membranes by the saponification of lipids. RNases catalyze RNA degradation. SV = Sample volume; BV = Buffer volume.
| ON-CHIP SAMPLE PREPARATION | |||||
|---|---|---|---|---|---|
| CHEMICAL LYSIS | |||||
| Sample Type | SV | BV | Reagents | Ref. | |
| Whole blood | 90 nL | n.a. | Sample droplet electrokinetically moved, mixed in a loop motion and incubated with Proteinase K and lysis buffer (details n.a.) | [22] | |
| 1 µL | 100 µL | Sample directly pipetted onto the extraction membrane. Water (200 µL) is aspirated over the sample and the membrane by the syringe pump. 10 mM NaOH (100 µL) is loaded into the extraction chamber and incubated for 5 min. 1 mM HCl (50 µL) is loaded to neutralize pH after lysis | [29] | ||
| 10 µL | 50 µL | Sample and lysis buffer (4 M GuSCN in TE; 1% Triton X-100; pH 6.7) simultaneously pumped from two inlet holes into the microchannel | [31] | ||
| 200 µL | n.a. | Sample mixed with lysis buffer, RNase-A, binding buffer (details n.a.) | [35] | ||
| 0.5 µL | 50 µL | Sample mixed into the reaction chamber with a lysis buffer solution (0.1% CTAB, 1.5 M NaCl, MES, pH 5.0) and incubated for 15 min at room temperature | [46] | ||
| 4 µL | 5.6 µL | Sample is diluted with PBS (if the blood is old), mixed with lysis buffer (5.6 µL), and incubated for 5 min | [49] | ||
| Plasma | 140 µL | 500 µL | Sample, DMP, lysis buffer (100 nM TrisHCl pH 8, 10 nM EDTA, 1% SDS, 10% Triton X-100), Proteinase K, DNase I (in case of RNA extraction) incubated for 20 min | [58] | |
| Saliva | 100 µL | 200 µL | Sample mixed with the cell lysis solution (Proteinase K and GuSCN-based lysis buffer) | [67] | |
| 100 µL | 100 µL | Sample mixed with pre-stored lysis and binding buffer (GuHCl-based) | [68] | ||
| 500 µL | 100 µL | Sample pipetted into loading chamber. Water (200 µL) is aspirated over the sample and the membrane by the syringe pump. 10mM NaOH (100 µL) is loaded into the extraction chamber and incubated for 5 min. 1 mM HCl (50 µL) is loaded to neutralize pH after lysis | [29] | ||
| Buccal swab | 100 µL | Swab is placed into loading chamber. Water (200 µL) is aspirated over the sample and the membrane by the syringe pump. 10 mM NaOH (100 µL) is loaded into the extraction chamber and incubated for 5 min. 1 mM HCl (50 µL) is loaded to neutralize pH after lysis | |||
| 20 µL | Swab incubated into the lysis solution (5 M GuHCl in 10 mM TE) at room temperature for 10 min | [65] | |||
| Semen | 2 µL 20 µL |
480 µL 496 µL |
Lysis for DNA extraction: semen (2 µL) diluted 1:1 with water, mixed with lysis buffer (496 µL, 6 M GuHCl with 40 mM DDT, pH 6.1) Lysis for RNA extraction: neat semen (20 µL) mixed with lysis buffer (480 µL, 6 M GuHCl with 40 mM DDT, pH 6.1) |
[9] | |
| 1 µL | n.a. | Sample loaded and mixed with lysis buffer (6 M GuHCl with 4 mM DDT) | [24] | ||
| Urine | 50 µL | 155 µL | Sample mixed with a lysis-binding solution containing a GuHCl-based lysis buffer (AL buffer, Qiagen), Proteinase K and DMA binding agent | [26] | |
| Stool | 400 µL | Liquid stool (diluted in water) mixed with pre-charged guanidine solid salts (reconstituting to 5 M GuHCl) and incubated for 5 min | [73] | ||
| MECHANICAL + CHEMICAL LYSIS | |||||
| Sample type | SV | BV | Mechanical step | Reagents | Ref. |
| Hematuria urine | 100 µL | 100 µL | Solution is pressure-forced (150 psi) through small pores polymer monolith | Sample is mixed with lysis solution containing Proteinase K (0.8 mg/mL), GuSCN and SDS (0.01%) | [75] |
| Whole blood, Urine | 2–10 µL | n.a. | Filtration | Sample is mixed with Proteinase K and AL Buffer, Qiagen | [52] |
| Whole blood | 1 µL | n.a. | Sample is premixed with PBS by the micromixer to adjust viscosity of the circulating solution. Sample flows through a pillar filtering structure of 3 µm spacing, which retains white blood cells (6–9 µm diameter) and lets other blood components pass through | Collected white blood cells are mixed with 6 M GuHCl | [39] |
| Nasal swab | 1 mL | Swab is priorly vortexed for 1 min to detach cells. Solution is loaded and the vibrating and flexible PDMS valve makes beads collide and cells to be captured, while providing a strong mixing | NaOH (6 µL; 0.02 N) is loaded for lysis | [68] | |
| Whole blood | 5 µL | 150 µL | Filtration with 3.5 µm pores structure to retain white blood cells and discard plasma and red blood cells | Blood in 0.9% NaCl (45 µL). Mixing white blood cells with loading buffer (1% Triton X-100 and 6 M GuSCN, pH 6.4) | [42] |
| Nasopharyngeal swab | 100 µL | 600 µL | Mixing occurs by air bubble insufflation | Sample is mixed with pre-treatment buffer (300 µL; PBS and Lysozyme) and with a GuSCN-based lysis buffer (300 µL). Incubated at room temperature for 3 min | [72] |
| Whole blood, serum, plasma | 200 µL | 600 µL300 µL | Constant mixing is provided by the control of rotational frequency | Sample is loaded with a lysis solution (600 µL; GuSCN or AL Buffer, Qiagen, Triton X-100, EDTA) or a GuHCl-based lysis buffer (300 µL; AL Buffer, Qiagen). Sample is incubated with lysis solutions for 15 min at room temperature | [28,64] |
| MECHANICAL + THERMAL LYSIS | |||||
| Sample type | SV | Mechanical step | Thermal step | Ref. | |
| Serum | 0.4 µL | Agitation | Irradiation (40 s) of the vibrating chamber with laser beam (808 nm) for heat shock | [23] | |
| CHEMICAL + THERMAL LYSIS | |||||
| Sample type | SV | BV | Reagents | Thermal step | Ref. |
| Nasopharyngeal swab | 500 µL | 500 µL | Sample is mixed with lysis buffer (500 µL; ML Buffer, Qiagen) and Proteinase K | Lysis chamber is heated with a resistive heater | [71] |
| Whole blood | 1 µL | 14 µL | Sample is mixed with lysis-electrolytic buffer (13 µL; 50 mM Tris, pH 8.2; 50 mN HEPES, 1 µL of Proteinase K) | Heating for 3 min with an on-chip resistive heater | [32] |
| THERMAL + MECHANICAL + CHEMICAL LYSIS | |||||
| Sample type | SV | BV | Thermal and mechanical steps | Reagents | Ref. |
| Whole blood | 5.4 µL | 5.8 µL | Chip heated and agitated by a magnet (56 °C for 6 min) | Sample is mixed with lysis buffer (5.4 µL, details n.a.) and Proteinase K (0.4 µL). | [34] |
| Whole blood | 300 µL | 330 µL | Heating with a thermoelectric heater (56 °C for 10 min). Mixing with an air bubble blow from the bottom of the chamber | Sample is mixed with Proteinase K (30 µL) and lysis buffer (300 µL; AL Buffer, Qiagen) | [36] |
| Stool | 200 mg | n.a. | Sample is heated (90 °C for 5 min) and homogenized by a small vibrating magnet. Homogenate mixed by air bubble insufflation and filtered by a 1 µm pore-size filter to remove fecal impurities | Lysis reagents (details n.a.) are mixed with sample by air pressure application. | [27] |