# Copper Annealing Temperature: Definition and PCR Role ## Introduction to Copper Annealing Temperature The term "copper annealing temperature" is a point of confusion for many students entering [molecular biology](/blog/careers/molecular-biology), and for good reason: the phrase juxtaposes a metallurgical concept with a nucleic acid chemistry concept. In the context of PCR ([polymerase chain reaction](/knowledge/molecular-biology/polymerase-chain-reaction)), **copper annealing temperature** does not refer to the heat treatment of copper metal. It is a colloquial—and somewhat misleading—shorthand that occasionally appears in laboratory protocols and student notes, where "copper" is a corruption or misremembering of the word "proper" or a confusion with the metal's annealing process. What the term actually designates is the **annealing temperature** used in a PCR thermal cycling protocol: the specific temperature at which oligonucleotide primers hybridize to their complementary sequences on single-stranded template DNA. To be absolutely clear: copper metal has an annealing temperature of approximately 200–400°C depending on the degree of cold working and the desired recrystallization outcome. This is a solid-state metallurgical process with zero relevance to PCR. When a protocol or textbook mentions "copper annealing temperature," it is either an error or a colloquialism for the primer annealing step temperature. Throughout this article, we will use the term as it is understood in the PCR context: the temperature at which primers anneal to template DNA during the [polymerase chain reaction](/knowledge/molecular-biology/polymerase-chain-reaction). The annealing step is the second phase of each PCR cycle, occurring after denaturation (typically 94–98°C) and before extension (typically 68–72°C). During annealing, the reaction temperature is lowered to allow the forward and reverse primers to base-pair with their complementary regions on the denatured, single-stranded template. The temperature chosen for this step is arguably the single most important variable in PCR optimization, as it directly governs the specificity and efficiency of amplification. A poorly chosen annealing temperature can produce no product, non-specific products, or primer-dimers, wasting time and reagents. ## The Role of Annealing Temperature in PCR The polymerase chain reaction, first conceptualized by Kary Mullis in 1983, relies on repeated cycles of denaturation, annealing, and extension to exponentially amplify a target DNA sequence. The annealing step is where the reaction's specificity is determined. Understanding the biophysics of this step is essential for designing robust PCR assays. ### Primer-Template Hybridization During annealing, the reaction mixture—containing template DNA, primers, deoxynucleotide triphosphates (dNTPs), buffer, and a thermostable [DNA polymerase](/blog/guides/dna-polymerase) such as *Taq* polymerase from *Thermus aquaticus*—is cooled from denaturation temperature to the annealing temperature, typically between 50°C and 65°C. At this temperature, the thermal energy is low enough that hydrogen bonds can form between complementary bases, yet high enough that mismatched hybrids are thermodynamically unstable. Primer-template hybridization is a reversible equilibrium process. The forward and reverse primers, typically 18–24 nucleotides in length, diffuse through the solution and collide with the single-stranded template. When a primer encounters a perfectly complementary region, it forms a stable duplex. The stability of this duplex depends on the cumulative strength of the hydrogen bonds (two for A-T pairs, three for G-C pairs) and the stacking interactions between adjacent bases. The temperature at which 50% of the primer molecules are bound to their target sequence is the melting temperature (Tm) of that primer-template duplex. The annealing temperature is always set below the Tm of the primers, typically 3–5°C lower. This ensures that a sufficient fraction of primer molecules are bound at any given moment for the polymerase to initiate extension. However, the exact offset depends on the primer sequence, buffer composition, and the polymerase's optimal activity temperature. ### Specificity vs. Efficiency Trade-off The central tension in choosing an annealing temperature is the balance between specificity and efficiency. At lower annealing temperatures (e.g., 45–50°C), primers bind more readily to their targets, increasing the amount of product synthesized per cycle. However, lower temperatures also permit primers to bind to partially complementary sequences elsewhere in the genome or in the reaction mixture. These off-target binding events can lead to non-specific amplification products, which appear as extra bands on an agarose gel and can compete with the desired product for reagents. At higher annealing temperatures (e.g., 60–65°C), the stringency increases: only perfectly matched primer-template duplexes are stable enough to persist. This dramatically reduces non-specific amplification but also reduces the efficiency of primer binding. If the annealing temperature is set too high—above the Tm of the primers—very few primer molecules will be bound at any time, and the reaction will yield little or no product. The optimal annealing temperature is therefore the highest temperature at which efficient, specific amplification still occurs. This is typically determined empirically, as we will discuss in the section on experimental determination. For most standard PCR reactions with well-designed primers, an annealing temperature of 55–60°C works well. For high-GC templates or primers with complex secondary structure, higher annealing temperatures may be required. ## Factors Influencing Optimal Annealing Temperature Several interrelated factors determine the ideal annealing temperature for a given primer pair. Understanding these variables allows you to predict a reasonable starting point and troubleshoot failed reactions. ### Primer Composition The most significant factor is the nucleotide composition of the primers themselves. Guanine and cytosine form three hydrogen bonds when paired, whereas adenine and thymine form only two. Consequently, primers with high GC content have higher melting temperatures and require higher annealing temperatures for optimal specificity. The GC content of a primer is calculated as the percentage of G and C nucleotides in the sequence. For example, a 20-nucleotide primer with 10 G/C bases has a GC content of 50%. Primers with 40–60% GC content are generally considered ideal for PCR. Primers with very high GC content (>70%) may form stable secondary structures such as hairpins or self-dimers, which can interfere with primer binding and reduce amplification efficiency. Conversely, primers with very low GC content (<40%) may have Tm values too low for practical annealing temperatures, requiring the use of lower extension temperatures or modified buffer conditions. The length of the primer also matters. Longer primers have more total hydrogen bonds and stacking interactions, giving them higher Tm values. A 24-mer with 50% GC content will have a higher Tm than an 18-mer with the same GC percentage. However, longer primers also have a higher probability of forming secondary structures and of containing regions of self-complementarity. ### Buffer Conditions The ionic strength of the PCR buffer significantly affects primer-template stability. Monovalent cations, particularly sodium (Na⁺) and potassium (K⁺), shield the negative charges on the phosphate backbones of DNA, reducing electrostatic repulsion between the two strands. Higher salt concentrations therefore stabilize duplex formation and increase the effective Tm of primers. Standard PCR buffers contain 50 mM KCl, which provides a moderate ionic strength. However, many commercial buffers now include proprietary additives. Some buffers contain ammonium sulfate ((NH₄)₂SO₄), which can improve specificity by differentially affecting matched versus mismatched duplex stability. Magnesium chloride (MgCl₂) is also present in PCR buffers at concentrations typically ranging from 1.5 to 3.0 mM. Magnesium is a required cofactor for DNA polymerase activity, but it also stabilizes primer-template duplexes. Higher Mg²⁺ concentrations increase the Tm of primers but can also promote non-specific binding. The optimal Mg²⁺ concentration is usually determined empirically for each primer pair and template combination. Some PCR additives, such as dimethyl sulfoxide (DMSO) or betaine, are used to reduce secondary structure in GC-rich templates. These additives lower the Tm of primers and template, so the annealing temperature may need to be reduced when they are used. ### Template Complexity The complexity of the template DNA also influences the optimal annealing temperature. Genomic DNA from higher eukaryotes, such as human DNA, contains approximately 3 billion base pairs with a high degree of sequence complexity. In such complex templates, the probability of a primer having a partial match somewhere in the genome is high. Higher annealing temperatures are therefore recommended for genomic DNA templates to reduce the likelihood of non-specific priming. In contrast, plasmid DNA or short amplicons (e.g., PCR products used for cloning or sequencing) have lower complexity, and lower annealing temperatures may be acceptable. Similarly, when amplifying from a cDNA library or a mixed microbial community, the annealing temperature should be adjusted based on the expected complexity of the template pool. ## Calculating Annealing Temperature: Formulas and Tools While empirical determination is the gold standard, several formulas and computational tools can provide a reasonable starting estimate for annealing temperature. These methods range from simple rule-of-thumb calculations to sophisticated thermodynamic models. ### Basic Formula The simplest and most widely taught formula is the Wallace rule, originally developed for predicting the melting temperature of short oligonucleotides used as hybridization probes: **Tm = 2(A + T) + 4(G + C)** In this formula, A, T, G, and C represent the number of each nucleotide in the primer. The annealing temperature is then typically set 5°C below the calculated Tm: **Ta = Tm − 5°C** For example, consider the primer sequence 5'-ATGCGTACGTAGCTAGCT-3'. This 18-mer contains 5 A, 5 T, 4 G, and 4 C. The Wallace rule gives: Tm = 2(5 + 5) + 4(4 + 4) = 20 + 32 = 52°C The suggested annealing temperature would be 47°C. The Wallace rule works reasonably well for primers shorter than 20 nucleotides but becomes increasingly inaccurate for longer primers. It also does not account for salt concentration or the sequence-dependent stacking interactions between adjacent bases. ### Nearest-Neighbor Thermodynamics A more accurate approach uses the nearest-neighbor model, which treats the stability of a DNA duplex as the sum of contributions from each adjacent base pair. This model accounts for the fact that the stability of a given base pair depends on its neighboring bases due to stacking interactions. The nearest-neighbor method calculates the Gibbs free energy change (ΔG) for duplex formation at a given temperature, using experimentally derived thermodynamic parameters for each of the 10 possible nearest-neighbor combinations (e.g., AA/TT, AT/TA, GC/CG, etc.). The melting temperature is then calculated from the enthalpy (ΔH) and entropy (ΔS) changes: **Tm = ΔH / (ΔS + R × ln(C_T/4))** where R is the gas constant (1.987 cal/(mol·K)) and C_T is the total concentration of the annealing oligonucleotide. This calculation is complex to perform by hand, which is why it is almost always implemented in software. The nearest-neighbor parameters used in most tools are derived from the work of John SantaLucia and colleagues, who compiled unified thermodynamic parameters for DNA duplex formation in 1 M NaCl. These parameters are then corrected for the actual salt concentration of the PCR buffer. ### Online Tools Several freely available online tools implement the nearest-neighbor model and provide reliable Tm and annealing temperature predictions. The most commonly used include: - **IDT OligoAnalyzer** (Integrated DNA Technologies): Provides Tm calculations using the nearest-neighbor model, with options to adjust salt and primer concentrations. - **Thermo Fisher Tm Calculator**: Designed specifically for PCR primers, this tool accounts for the buffer conditions of Thermo Fisher's PCR master mixes. - **NEB Tm Calculator** (New England Biolabs): Similar to the Thermo Fisher tool but optimized for NEB polymerases and buffers. - **Primer3**: A primer design program that calculates Tm and annealing temperatures as part of its primer selection algorithm. These tools typically ask for the primer sequence, the concentration of monovalent cations (usually 50 mM for standard PCR buffer), the concentration of free magnesium (often 1.5–3.0 mM), and the primer concentration (typically 0.2–0.5 μM). The output is a predicted Tm, and the recommended annealing temperature is usually Tm minus 3–5°C. It is important to note that different tools may give slightly different Tm values for the same primer because they use different thermodynamic parameter sets or salt correction formulas. This is normal and underscores the need for empirical optimization. ## Experimental Determination of Optimal Annealing Temperature While calculations provide a useful starting point, the only way to know the true optimal annealing temperature for a given primer pair is to test it experimentally. Two common approaches are gradient PCR and touchdown PCR. ### Gradient PCR Gradient PCR is a straightforward method for determining the optimal annealing temperature in a single experiment. A thermal cycler with gradient capability heats the block in a controlled temperature gradient, so that different columns or rows of wells are maintained at different temperatures simultaneously. For example, you might set the gradient to span from 50°C to 65°C across a 12-column block, with each column at a distinct temperature. The experimental setup is simple: 1. Prepare a single PCR master mix containing template, primers, dNTPs, buffer, and polymerase. 2. Aliquot equal volumes into each well of the PCR plate or strip tubes. 3. Program the thermal cycler with a gradient annealing step spanning your predicted range. 4. Run the PCR for the desired number of cycles (typically 30–35). 5. Analyze the products by agarose gel electrophoresis. The optimal annealing temperature is the lowest temperature at which you see a single, bright, correctly sized band with no non-specific products. If all temperatures produce non-specific bands, the entire gradient may need to be shifted higher. If no temperatures produce product, the gradient should be shifted lower or the reaction conditions (e.g., Mg²⁺ concentration) should be adjusted. ### Touchdown PCR Touchdown PCR is an alternative strategy that does not require a gradient-capable thermal cycler. Instead, the annealing temperature is progressively decreased by 1°C per cycle over the first several cycles, then held constant for the remaining cycles. A typical touchdown protocol might be: 1. Initial denaturation: 95°C for 2–3 minutes. 2. Touchdown phase (10 cycles): Annealing temperature starts at 65°C and decreases by 1°C per cycle (65°C, 64°C, 63°C, ... down to 56°C). 3. Amplification phase (20–25 cycles): Annealing temperature held at 55°C. 4. Final extension: 72°C for 5–10 minutes. The logic behind touchdown PCR is that the initial high annealing temperatures allow only perfectly matched primer-template duplexes to form, enriching the reaction for the correct product. As the temperature decreases in subsequent cycles, the correct product is already being amplified and will outcompete any non-specific products that might form at lower temperatures. Touchdown PCR is particularly useful when the optimal annealing temperature is unknown or when amplifying from complex templates. ## Common Mistakes and Misconceptions Students and even experienced researchers frequently make errors when working with annealing temperatures. Recognizing these pitfalls can save significant time and frustration. ### Annealing vs. Melting Temperature The most common confusion is between annealing temperature (Ta) and melting temperature (Tm). These are related but distinct concepts. The melting temperature is the temperature at which 50% of a duplex DNA population is dissociated into single strands. It is a property of the primer-template duplex under specific buffer conditions. The annealing temperature is the temperature at which the PCR thermal cycler is set for the primer-binding step. It is always lower than the Tm, typically by 3–5°C, but sometimes by as much as 10°C for high-stringency reactions. Some students mistakenly set the annealing temperature equal to the Tm, which results in very little primer binding and poor amplification. Others set it far below the Tm, which maximizes binding but sacrifices specificity. The relationship between these two temperatures is discussed in more detail in our article on __MASK_1__. ### Ignoring Salt Concentrations As discussed earlier, the Tm of a primer depends on the ionic strength of the solution. The Wallace rule and many basic calculations assume standard salt conditions (approximately 50 mM monovalent cations). If you are using a buffer with different salt concentrations—for example, a high-salt buffer for GC-rich templates or a buffer with added ammonium sulfate—the actual Tm will differ from the calculated value. Many commercial PCR buffers now contain proprietary salt formulations that differ significantly from the traditional 50 mM KCl. The Tm calculators provided by the buffer manufacturer are often the most accurate for their specific products. Ignoring salt effects can lead to annealing temperatures that are several degrees off from optimal, resulting in weak or non-specific amplification. ### Using Wrong Formula The Wallace rule is appropriate for short oligonucleotides (14–20 nucleotides) but becomes increasingly inaccurate for longer primers. For primers longer than 20 nucleotides, the nearest-neighbor method is strongly preferred. Additionally, the Wallace rule assumes a GC content that is not too extreme; for primers with very high or very low GC content, even the nearest-neighbor method may need correction. Another common error is using a formula that does not account for the primer concentration. The Tm of a primer depends on its concentration: higher primer concentrations stabilize the duplex and increase the apparent Tm. Most PCR reactions use primer concentrations of 0.2–0.5 μM, and the nearest-neighbor calculations should be performed at the actual primer concentration used in the reaction. ### Confusing Annealing with Extension Some students mistakenly believe that the annealing temperature is also the temperature at which the polymerase extends the primer. This is incorrect. In a standard three-step PCR, annealing and extension are separate steps with different temperatures. The annealing temperature is typically 50–65°C, while the extension temperature is 68–72°C (the optimal activity temperature for *Taq* polymerase). Some polymerases, such as those engineered for fast PCR, can perform both annealing and extension at the same temperature (a two-step PCR protocol), but this is a specialized approach. ## Practical Tips for Setting Annealing Temperature Armed with an understanding of the theory, you can now approach annealing temperature selection systematically. ### Starting Point For a newly designed primer pair, begin with the following steps: 1. Use a reputable Tm calculator (e.g., IDT OligoAnalyzer, NEB Tm Calculator) to predict the Tm of each primer. 2. If the two primers have different Tm values, use the lower Tm as the basis for your annealing temperature calculation. 3. Set the initial annealing temperature to 3–5°C below the lower Tm. 4. Run a gradient PCR spanning ±5°C around this initial estimate to empirically determine the optimal temperature. For example, if your forward primer has a predicted Tm of 60°C and your reverse primer has a predicted Tm of 58°C, start with an annealing temperature of 53–55°C and run a gradient from 50°C to 60°C. ### Adjusting for Specificity If you observe non-specific bands in your PCR product, increase the annealing temperature in 1–2°C increments. Each increase will reduce non-specific binding while potentially decreasing yield. Continue increasing until the non-specific bands disappear while the desired band remains bright. If you observe no product at all, first verify that your primers are correct and that the template is intact. Then decrease the annealing temperature in 1–2°C increments. If you still see no product at temperatures 10°C below the predicted Tm, consider other factors such as primer design, template quality, or polymerase activity. ### Troubleshooting When PCR fails, the annealing temperature is a prime suspect, but it is not the only variable to consider. A systematic troubleshooting approach is essential: - **No product**: Check template concentration and quality, primer integrity, polymerase activity, and extension time. If all are adequate, lower the annealing temperature. - **Multiple bands**: Increase annealing temperature, reduce primer concentration, or increase Mg²⁺ concentration (which can paradoxically improve specificity in some cases by stabilizing the correct product). - **Smear on gel**: This often indicates excessive template or too many cycles. Reduce cycle number or template concentration. Alternatively, the annealing temperature may be too low, allowing non-specific priming throughout the reaction. - **Primer-dimers**: These appear as low-molecular-weight bands and result from primers annealing to each other. Increase annealing temperature, redesign primers to avoid complementarity at the 3' ends, or reduce primer concentration. For additional guidance on finding the right annealing temperature, see our article on how to __MASK_2__. If you are working with templates that are difficult to denature, you may also need to consider how __MASK_3__ stability during the denaturation step. ## Summary and Key Takeaways The annealing temperature is a critical parameter in PCR that determines the specificity and efficiency of primer binding to template DNA. Despite the confusing terminology, "copper annealing temperature" in a PCR context refers to the primer annealing step temperature, not to any property of copper metal. The optimal annealing temperature depends on primer length and GC content, buffer salt composition, template complexity, and the specific polymerase used. It can be estimated using formulas such as the Wallace rule or, more accurately, the nearest-neighbor thermodynamic model implemented in online calculators. However, empirical determination through gradient PCR or touchdown PCR is the most reliable approach. Common mistakes include confusing annealing temperature with melting temperature, ignoring salt effects on Tm, using inappropriate formulas for primer length, and failing to distinguish annealing from extension steps. By understanding the underlying biophysics and following systematic optimization strategies, you can reliably set annealing temperatures that produce clean, specific, and efficient PCR amplification. ## Frequently Asked Questions ### What is the annealing temperature of copper? In metallurgy, copper annealing is a heat treatment process performed at approximately 200–400°C to soften work-hardened copper and restore its ductility. However, in the context of PCR and molecular biology, "copper annealing temperature" is a misnomer or colloquialism that refers to the primer annealing temperature—the temperature at which oligonucleotide primers hybridize to template DNA during PCR. This temperature is typically 50–65°C and has nothing to do with the metal copper. ### How do I calculate the annealing temperature for my primers? The simplest method is the Wallace rule: Tm = 2(A + T) + 4(G + C), with the annealing temperature set 5°C below Tm. For more accurate results, use a nearest-neighbor thermodynamic calculator such as IDT OligoAnalyzer or the NEB Tm Calculator, which account for salt concentration, primer concentration, and sequence-dependent stacking interactions. Always verify the calculated temperature empirically with a gradient PCR. ### What is the difference between annealing temperature and melting temperature? The melting temperature (Tm) is the temperature at which 50% of a primer-template duplex population is denatured into single strands. It is a physical property of the duplex under specific buffer conditions. The annealing temperature (Ta) is the temperature set on the thermal cycler for the primer-binding step of PCR. Ta is always lower than Tm, typically by 3–5°C, to allow sufficient primer binding while maintaining specificity. For a deeper comparison, see __MASK_4__. ### Why is my PCR not working? Could it be the annealing temperature? Yes, an incorrect annealing temperature is a common cause of PCR failure. If the annealing temperature is too high, primers will not bind efficiently, and no product will be produced. If it is too low, non-specific products and primer-dimers may form. However, PCR failure can also result from degraded template, inactive polymerase, incorrect Mg²⁺ concentration, or poor primer design. Run a gradient PCR to test a range of annealing temperatures, and systematically troubleshoot other variables if the problem persists. ### Does the annealing temperature depend on the DNA polymerase used? Yes, to some extent. Different DNA polymerases have different optimal activity temperatures and buffer requirements. For example, *Taq* polymerase works well at 68–72°C for extension and tolerates annealing temperatures of 50–65°C. High-fidelity polymerases such as Phusion or Q5 have optimal extension temperatures around 72°C and often require higher annealing temperatures due to their proprietary buffer formulations. Some engineered polymerases are designed for two-step PCR, where annealing and extension occur at the same temperature (typically 60–68°C). Always consult the manufacturer's recommendations for the specific polymerase you are using. ### What is the typical annealing temperature for PCR? For most standard PCR reactions with well-designed primers (18–24 nucleotides, 40–60% GC content), the typical annealing temperature is 55–60°C. However, this can range from as low as 45°C for AT-rich primers to as high as 68°C for GC-rich primers or when using high-stringency conditions. The optimal temperature for your specific primers should be determined empirically. ### How does GC content affect annealing temperature? Guanine and cytosine form three hydrogen bonds in a base pair, whereas adenine and thymine form only two. Therefore, primers with higher GC content form more stable duplexes with the template and have higher melting temperatures. This means they require higher annealing temperatures for optimal specificity. As a rule of thumb, each 1% increase in GC content raises the Tm by approximately 0.4°C for primers in the 18–24 nucleotide range. Primers with very high GC content (>70%) may also form secondary structures that interfere with annealing and may require additives such as DMSO or betaine to improve amplification. ## Key Takeaways - "Copper annealing temperature" in PCR refers to the primer annealing temperature, not to the metallurgical annealing of copper metal. - The annealing temperature is the thermal cycler setting at which primers bind to template DNA, typically 3–5°C below the primer melting temperature (Tm). - Annealing temperature controls the trade-off between PCR specificity and efficiency: higher temperatures increase specificity but reduce yield, while lower temperatures do the opposite. - Primer length, GC content, buffer salt concentration, and template complexity all influence the optimal annealing temperature. - Use the Wallace rule for quick estimates with short primers, but prefer nearest-neighbor thermodynamic calculators for accurate predictions with longer primers. - Gradient PCR and [touchdown PCR](/knowledge/diagnostics/molecular/touchdown-pcr-reducing-nonspecific-amplification) are reliable experimental methods for determining the optimal annealing temperature empirically. - Common mistakes include confusing Ta with Tm, ignoring salt effects, and using inappropriate formulas for primer length. - For a comprehensive understanding of the broader PCR process, review the fundamentals of the [Polymerase Chain Reaction](/knowledge/molecular-biology/polymerase-chain-reaction) and the detailed [PCR Explained](/knowledge/molecular-biology/pcr-explained) guide. ## Further Reading - Liu Y et al. *Effects of Annealing Temperature on Interfacial Structure and Thermal Conductivity of Hot-Pressed Copper/Cr-Coated Diamond Composites*. Materials (Basel, Switzerland). 2026. [PubMed 42073701](https://doi.org/10.3390/ma19081534) - Kim GH et al. *Effects of Annealing Temperature on the Oxygen Evolution Reaction Activity of Copper-Cobalt Oxide Nanosheets*. Nanomaterials (Basel, Switzerland). 2021. [PubMed 33800286](https://doi.org/10.3390/nano11030657) - Tseng JY, Chen WJ, Chen PH. *The Impact of Substrate Temperature on the Adhesion Strength of Electroplated Copper on an Al-Doped ZnO/Si System*. Materials (Basel, Switzerland). 2024. [PubMed 39459657](https://doi.org/10.3390/ma17204953) - Qin J et al. *Microstructure and Texture Evolution in Cold-Rolled and Annealed Oxygen-Free Copper Sheets*. Materials (Basel, Switzerland). 2024. [PubMed 38793268](https://doi.org/10.3390/ma17102202) - Jin Y et al. *Effect of Post-Weld Annealing on Microstructure and Growth Behavior of Copper/Aluminum Friction Stir Welded Joint*. Materials (Basel, Switzerland). 2020. [PubMed 33076491](https://doi.org/10.3390/ma13204591) - Yahyaabadi A et al. *Effect of annealing temperature and dopant concentration on the thermoluminescence sensitivity in LiF:Mg,Cu,Ag material*. Luminescence : the journal of biological and chemical luminescence. 2018. [PubMed 29687582](https://doi.org/10.1002/bio.3487)
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