The Key to Lyophilized Products—Consistent Performance
How Lyophilized Products Improve Sensitivity in Diagnostic Assays
In diagnostics, sensitivity often comes down to one significant criterion: consistency. Even with a solid assay design, performance can drift because reagents behave differently over time, or across storage conditions. Lyophilization, the simple process of removing water to lock the molecular components into a dry, stable form, solves much of that. By minimizing run-to-run and long-term variance, lyophilization effectively sharpens assay resolution, allowing smaller true signals to stand out from noise and increasing overall sensitivity. It’s the quiet backbone behind modern PCR and immunoassay kits that deliver repeatable, high-sensitivity results.
Stabilizing What Matters
Enzymes, antibodies, and cofactors rarely stay perfectly stable in liquid formats. Slight temperature fluctuations during shipping or storage can change their activity. However, once freeze-dried, reagents tend to stay exactly as formulated. For example, Taq and reverse transcriptase enzymes in multiplex PCR lyophilized products can sit at room temperature for months and years yet rehydrate to perform identically to fresh mixes. (Kumar et al. 2020, Yang and Wen 2021, Xu et al. 2021). That kind of long-term stability is what allows assays to maintain high sensitivity without relying on a strict cold chain.
Lower Background, Clearer Signal
Lyophilization doesn’t remove salts or additives from the formulation, but what it does, is lock reagents into a uniform, highly stable matrix. Once rehydrated, that uniformity leads to more consistent visual signal responses. Additionally, fluorescence intensity in many cases improved, dependent on assay platform (Thirion et al. 2020, Hammerling et al. 2021). In effect, the assay behaves as though the background noise has dropped - not because contaminants are gone, but because every component is fixed in place and reconstitutes predictably. The result is improved signal-to-noise and fewer false readings across runs.
More Consistent Runs
Another benefit most labs notice right away is tighter control data. Lyophilized products such as beads are pre-measured and sealed, so each test starts with the same baseline composition. That eliminates small pipetting or mixing variations that can affect threshold cycles or endpoint readings. Variability between operators or batches is reduced.
Supporting Complex Assays
Freeze-drying also makes multiplex and multi-step assays feasible and more practical. You can pre-load beads with enzyme, buffer, and probe combinations that would otherwise be unstable, or incompatible in liquid form. Many of today’s UTI and STI molecular panels rely on this approach, each lyophilized bead delivers a fixed reagent set for its specific target, enabling consistent amplification across multiple pathogens in one run.
Stronger Signal Amplification
Amplification-based methods like PCR and LAMP can benefit the most from lyophilized products. Lyophilized primers, probes, and polymerases hold their conformation through storage, producing cleaner, steeper amplification curves. In several studies, including RT-LAMP assays for Zika, influenza A, and SARS-CoV-2, lyophilized reaction pellets maintained full enzyme activity after storage and often showed improvement in detection limit compared with their liquid counterparts (Prado et al. 2024, Moore et al. 2021, Matl et al. 2025). When working at the edge of sensitivity, that kind of improvement can mean the difference between early detection and a missed case.
Getting the Process Right
The real performance gain comes from process control. The freezing process, the freeze-drying cycle, and the choice of exipients must balance temperature, pressure, and time so that reagents dry fully without denaturing. Adding the right protectants, trehalose, mannitol, or small amino acids, prevents collapse and preserves enzyme structure. Regular QC of residual moisture and rehydration performance keeps each batch consistent. It’s not glamorous work but it is what delivers assay consistency.
Summary:
In Practice
Lyophilized products will not make an assay inherently better, but it makes the assay more consistent. Stable reagents behave predictably, which means cleaner signals and fewer repeat tests. This consistency delivers assays that won’t lose sensitivity from storage or transportation, unlike their liquid brethren.
References:
Kumar S, Gallagher R, Bishop J, Kline E, Buser J, Lafleur L, Shah K, Lutz B, Yager P. Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices. Analyst. 2020 Oct 26;145(21):6875-6886.
Yang S, Wen W. Lyophilized Ready-to-Use Mix for the Real-Time Polymerase Chain Reaction Diagnosis. ACS Appl Bio Mater. 2021 May 17;4(5):4354-4360.
Jin J, Zeng Y, Gao X, Li J, Cui T, Xu X, Yang G, Zhang G, Hao C, Zhang J. Trehalose and mannitol based lyoprotection of Taq DNA polymerase for cold-chain-free long-term storage. J Pharm Sci. 2025 May;114(5):103656.
Xu J, Wang J, Su X, Qiu G, Zhong Q, Li T, Zhang D, Zhang S, He S, Ge S, Zhang J, Xia N. Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics. Talanta. 2021 Dec 1;235:122797.
Thirion L, Dubot-Peres A, Pezzi L, Corcostegui I, Touinssi M, de Lamballerie X, Charrel RN. Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solution for Standardization of Real-Time PCR and RT-qPCR Diagnostics in Virology. Viruses. 2020 Jan 30;12(2):159.
Hammerling MJ, Warfel KF, Jewett MC. Lyophilization of premixed COVID-19 diagnostic RT-qPCR reactions enables stable long-term storage at elevated temperature. Biotechnol J. 2021 Jul;16(7):e2000572.
Prado, N.O., Marin, A.M., Lalli, L.A. et al. Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19. Sci Rep 14, 10612 (2024).
Moore KJM, Cahill J, Aidelberg G, Aronoff R, Bektaş A, Bezdan D, Butler DJ, Chittur SV, Codyre M, Federici F, Tanner NA, Tighe SW, True R, Ware SB, Wyllie AL, Afshin EE, Bendesky A, Chang CB, Dela Rosa R 2nd, Elhaik E, Erickson D, Goldsborough AS, Grills G, Hadasch K, Hayden A, Her SY, Karl JA, Kim CH, Kriegel AJ, Kunstman T, Landau Z, Land K, Langhorst BW, Lindner AB, Mayer BE, McLaughlin LA, McLaughlin MT, Molloy J, Mozsary C, Nadler JL, D'Silva M, Ng D, O'Connor DH, Ongerth JE, Osuolale O, Pinharanda A, Plenker D, Ranjan R, Rosbash M, Rotem A, Segarra J, Schürer S, Sherrill-Mix S, Solo-Gabriele H, To S, Vogt MC, Yu AD, Mason CE; gLAMP Consortium. Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications. J Biomol Tech. 2021 Sep;32(3):228-275.
Matl M, Kellner MJ, Ansah F, Grishkovskaya I, Handler D, Heinen R, Bauer B, Menéndez-Arias L, Auer TO, Prieto-Godino LL, Penninger JM; Vienna Covid-19 Detection Initiative (VCDI); Awandare GA, Brennecke J, Pauli A. A lyophilized open-source RT-LAMP assay for molecular diagnostics in resource-limited settings. Life Sci Alliance. 2025 Jul 14;8(10):e202403167.
