Lyophilized pellets
Lyophilized pellets, suitable for use in a microfluidic device, and a method for preparing the same are described. The lyophilized pellets contain various biological reagents, or microparticles, and a cryoprotectant. The lyophilized pellets have a high degree of sphericity and are in the size range 0.5 to 35 μL. The pellets are prepared by dispensing drops of reagent solution on to a cryogenically cooled plate, followed by subjecting to a vacuum.
1 . A lyophilized pellet, comprising:
a cryoprotectant;
a PCR reagent mix;
a salt selected from the group consisting of: KCl, MgCl 2 , and (NH 4 ) 2 SO 4 ; and
optionally a buffering agent;
wherein the lyophilized pellet has a volume in the range 0.5 to 35 μL.
2 . A lyophilized pellet according to claim 1 , wherein the PCT reagent mix comprises:
at least one enzyme;
at least one protein;
at least one primer;
at least one fluorogenic probe;
at least one plasmid;
at least one polypeptide; and
at least one nucleotide selected from the group consisting of: dATP, dGTP, dCTP, dTTP, and dUTP; and
optionally at least one non-specific control nucleic acid.
3 . A lyophilized pellet according to claim 1 , having a buffering agent, wherein the buffering agent is Tris.
4 . A lyophilized pellet according to claim 1 , wherein the cryoprotectant is trehalose.
5 . A lyophilized pellet according to claim 1 , additionally comprising a bulking agent.
6 . A method for making a lyophilized pellet, comprising:
introducing a liquid composition into a dispensing tip;
positioning the dispensing tip above a cryogenically cooled plate, wherein the plate has a hydrophobic surface, and wherein the tip is in close proximity to the surface;
dispensing a droplet of the liquid composition from the tip, on to the surface, wherein the droplet is momentarily in contact with both the tip and the surface;
removing the tip away from close proximity to the surface so that the droplet remains in contact with the surface;
maintaining the droplet in contact with the surface for such time as the droplet freezes to form a frozen droplet; and
placing the frozen droplet into a lyophilizer under conditions sufficient to produce a lyophilized pellet.
7 . The method of claim 6 wherein the cryogenically cooled plate has a temperature of about −65° C. to −180° C.
8 . The method of claim 6 wherein the hydrophobic surface is essentially flat.
9 . The method of claim 6 wherein the hydrophobic surface is selected from the group consisting of: a diamond surface; a silicon oxide surface; a diamond-SiO 2 slide; and PTFE.
10 . The method of claim 6 wherein said method is multiplexed, comprising a number of dispensing tips, thereby producing a number of lyophilized pellets simultaneously.
11 . The method of claim 6 wherein the lyophilized pellet is essentially spherical.
12 . The method of claim 6 wherein the liquid composition comprises a concentration of microspheres.
13 . The method of claim 12 wherein the concentration is in the range of 10 3 to 10 13 microspheres per mL.
14 . The method of claim 12 wherein the concentration is 10 6 -10 8 microspheres per mL.
15 . A lyophilized pellet according to claim 12 , containing between about 1 and about 10 12 microspheres.
16 . The method of claim 6 wherein the microspheres are coated with a polycationic material.
17 . The method of claim 6 wherein the liquid composition comprises a mixture of biological reagents selected from the group consisting of: a mixture of enzymes; and a PCR reagent mix.
18 . The method of claim 6 wherein close proximity to the surface is between 0.5 and 1.5 mm.
19 . The method of claim 6 wherein the lyophilized pellet has a volume between 0.5 and 35 μL.
20 . The method of claim 6 wherein the lyophilized pellet has a volume between 2 and 25 μL.
21 . The method of claim 6 wherein the lyophilized pellet has a volume between 1 and 10 μL.
22 . The method of claim 6 , wherein the conditions sufficient to produce a lyophilized pellet include a period of residency in the lyophilizer from about 20 to 40 hours.
23 . The method of claim 6 wherein the lyophilized pellet has a diameter between about 0.5 and about 5 mm.
24 . A lyophilized pellet made by the method of claim 6 having a sphericity between 0.75 and 1.
25 . An apparatus for preparing lyophilized pellets, comprising:
a cryogenically cooled plate having a hydrophobic surface;
a dispensing system configured to position a dispensing tip above and in proximity to the hydrophobic surface, wherein the dispensing tip is configured to dispense a droplet of liquid onto the hydrophobic surface causing a frozen droplet to form; and
a chamber enclosing at least the hydrophobic surface, configured to apply conditions of temperature and pressure sufficient to lyophilize the frozen droplet.
26 . A microfluidic cartridge, comprising:
a reagent inlet, wherein are situated one or more lyophilized pellets that each contain one or more reagents; and
a microfluidic network, having at least one microfluidic channel in communication with the reagent inlet, wherein the channel is configured to permit fluid to pass from the inlet into the channel;
wherein the one or more lyophilized pellets have a composition according to claim 1 .
27 . The cartridge of claim 26 , further comprising:
a lysis chamber, wherein are situated one or more lyophilized pellets that each contain one or more lysis reagents.
28 . The cartridge of claim 26 , wherein the network further comprises at least one component selected from the group consisting of: a valve; a gate; an outlet; a vent; and a channel.
29 . A lyophilized pellet, comprising:
a cryoprotectant; and
a plurality of microspheres having a concentration in the range of 10 3 to 10 13 per mL;
wherein the lyophilized pellet has a volume in the range 0.5 to 35 μL.
30 . A lyophilized pellet according to claim 29 , having an essentially spherical shape.
31 . A lyophilized pellet according to claim 29 wherein the microspheres are coated with a polycationic material.
32 . A lyophilized pellet according to claim 31 , wherein the polycationic material is selected from the group consisting of: poly-D-lysine; polyethyleneimine; poly-DL-ornithine; and poly-histidine.
33 . A lyophilized pellet according to claim 32 wherein the polycationic material is poly-D-lysine whose constituent molecules have molecular weights between 1,000 and 4,000 Daltons.
34 . A lyophilized pellet according to claim 33 , wherein the poly-D-lysine has an average molecular weight of about 1770 Daltons.
35 . A lyophilized pellet according to claim 32 wherein the polycationic material is polyethyleneimine whose constituent molecules have molecular weights between 600 and 800 Daltons.
36 . A lyophilized pellet according to claim 32 wherein the polycationic material is poly-DL-ornithine whose constituent molecules have molecular weights between 12,000 and 30,000 Daltons.
37 . A lyophilized pellet according to claim 29 , additionally comprising a bulking agent.
38 . A lyophilized pellet according to claim 29 , wherein the cryoprotectant is trehalose.
39 . A lyophilized pellet according to claim 29 , additionally comprising a mixture of enzymes.
40 . A lyophilized pellet, comprising:
a cryoprotectant;
a primer;
a probe;
an internal control plasmid;
a specificity control;
a PCR reagent;
optionally a salt;
optionally a bulking agent; and
a polymerase.
41 . A lyophilized pellet according to claim 39 , wherein:
the primer, the probe, and the specificity control, are specific to Group B Streptococcus.
42 . A lyophilized pellet according to claim 39 , wherein:
the primer, the probe, and the specificity control, are specific to a pathogen selected from the group consisting of: Yersinia pestis; Erwinia herbicola; Bacillus anthracis; Bacillus globigii; Listeria monocytogenes; E. coli O157; Herpes Simplex Virus 1; and Herpes Simplex Virus 2.