Accuracy in Microvolume Measurements. Investigating the Precision and Reproducibility of Cary 50, 300 and 4000 series Instruments for Microlitre samples

Importance of the topic

The accurate measurement and quantitation of DNA and biomolecules in microvolume samples are crucial for modern molecular biology, diagnostics, and biopharmaceutical research. Advances in UV/Vis spectrophotometry that allow reliable readings in volumes below 5 µL address sample scarcity, reduce reagent costs, and enable kinetic assays in confined formats.

Objectives and study overview

This study evaluates the precision, sensitivity, and reproducibility of three Cary series UV/Vis spectrophotometers (Cary 50, Cary 300, Cary 4000) paired with a Hellma TrayCell microvolume accessory. The aim is to define detection limits for single-strand DNA, compare platform performance, and assess suitability for low-volume quantitation and kinetic measurements.

Methodology and Instrumentation

• Alignment of microvolume TrayCell using built-in software modules and mechanical adjustments.
• Measurement of DNA at concentrations ranging from 0.5 to 50 µg/µL in 1 mm or 0.2 mm pathlength cells.
• Evaluation of repeatability by removal/replacement of the microcell and repeated scanning.
• Comparison of baseline noise, dynamic range, and evaporation resistance across instruments.

Key results and discussion

• Cary 50 achieved reliable DNA quantitation down to 2–5 ng/µL with quantitation error of ~3% at 5 µg/µL; however, scan noise approached 20%.
• Cary 300 provided enhanced dynamic range (up to 4 Abs in 1 mm cells) and maintained precision within <1% variance despite only 6% transmission through the TrayCell.
• Cary 4000 delivered the lowest baseline noise (0.0001 Abs) and extended quantitation to 0.5 µg/µL ssDNA with ~10% error, enabling sub-nanogram measurements.
• All instruments resisted significant drift or evaporation over multi-scan intervals, supporting kinetic assays in microvolumes.

Benefits and practical applications

• Enables accurate DNA and protein assays in precious or limited samples.
• Facilitates rapid quality control in biotech and clinical settings.
• Supports microvolume enzyme kinetics and real-time reaction monitoring.
• Reduces consumable costs by minimizing required sample volume.

Future trends and opportunities

Emerging developments may include integrated microfluidic spectrophotometers for automated sample handling, enhanced fiber-optic coupling to further boost light throughput, and software algorithms for real-time spectral deconvolution in sub-microliter assays. Integration with machine-learning models could optimize detection limits and compensate for noise in ultra-dilute samples.

Conclusion

The comparative study demonstrates that while the Cary 4000 offers superior sensitivity and stability for sub-microliter measurements, the Cary 50 and Cary 300 remain cost-effective and robust options for routine microvolume quantitation. Selection should consider required detection limits, sample throughput, and budget constraints.

Instrumentation used

• Cary 50 UV/Vis Spectrophotometer with Xenon flash lamp
• Cary 300 Research Grade UV/Vis Spectrophotometer
• Cary 4000 True Double Monochromator UV/Vis Spectrophotometer
• Hellma TrayCell microvolume accessory